Transposon, gene transfer system and method of using the same

ABSTRACT

The present invention refers to hyperactive variants of a transposase of the transposon system Sleeping Beauty (SB). The invention further refers to corresponding nucleic acids producing these variants, to a gene transfer system for stably introducing nucleic acid(s) into the DNA of a cell by using these hyperactive variants of a transposase of the transposon system Sleeping Beauty (SB) and to transposons used in the inventive gene transfer system, comprising a nucleic acid sequence with flanking repeats (IRs and/or RSDs). Furthermore, applications of these transposase variants, the transposon, or the gene transfer system are also disclosed such as gene therapy, insertional mutagenesis, gene discovery (including genome mapping), mobilization of genes, library screening, or functional analysis of genomes in vivo and in vitro. Finally, pharmaceutical compositions and kits are also encompassed.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a division of U.S. Ser. No. 14/957,877, filedDec. 3, 2015, now U.S. Pat. No. 9,840,696, which is a division of U.S.Ser. No. 12/667,527, filed Jun. 28, 2010, now U.S. Pat. No. 9,228,180,which is a National Stage Application claiming the priority ofco-pending PCT Application No. PCT/EP2008/005342, filed Jun. 30, 2008which in turn claims the benefit of priority to European Application No.07013109.9, filed Jul. 4, 2007 and European Application No. 07016202.9,filed Aug. 17, 2007. Applicant claims the benefits of both 35 U.S.C. §119 and 35 U.S.C. § 120 as to the PCT application, and the entiredisclosures of all of the referenced applications are incorporatedherein by reference in their entireties.

The present invention refers to hyperactive variants of a transposase ofthe transposon system Sleeping Beauty (SB). The invention further refersto corresponding nucleic acids producing these variants, to a genetransfer system for stably introducing nucleic acid(s) into the DNA of acell by using these hyperactive variants of a transposase of thetransposon system Sleeping Beauty (SB) and to transposons used in theinventive gene transfer system, comprising a nucleic acid sequence withflanking repeats (IRs and/or RSDs). Furthermore, applications of thesetransposase variants or the gene transfer system are also disclosed suchas gene therapy, insertional mutagenesis, gene discovery (includinggenome mapping), mobilization of genes, library screening, or functionalanalysis of genomes in vivo and in vitro. Finally, pharmaceuticalcompositions and kits are also encompassed.

In the era of functional genomics, there is a sore need for developingefficient means to explore the roles of genes in different cellularfunctions and, if necessary, to provide effective means for adequatelymodulating these genes in vitro and in vivo. Such methods, apart fromothers, particularly comprise methods for introducing DNA into a cell.

Typical methods for introducing DNA into a cell include DNA condensingreagents such as calcium phosphate, polyethylene glycol, and the like,lipid-containing reagents, such as liposomes, multi-lamellar vesicles,and the like, as well as virus-mediated strategies. However, all ofthese methods have their limitations. For example, there are sizeconstraints associated with DNA condensing reagents and virus-mediatedstrategies. Further, the amount of nucleic acid that can be transfectedinto a cell is limited in virus strategies. Not all methods facilitateinsertion of the delivered nucleic acid into cellular nucleic acid andwhile DNA condensing methods and lipid-containing reagents arerelatively easy to prepare, the insertion of nucleic acid into viralvectors can be labor intensive. Moreover, virus-mediated strategies canbe cell-type or tissue-type specific and the use of virus-mediatedstrategies can create immunologic problems when used in vivo.

One suitable tool in order to overcome these problems are transposons.Transposons or transposable elements include a (short) nucleic acidsequence with terminal repeat sequences upstream and downstream thereof.Active transposons encode enzymes that facilitate the excision andinsertion of the nucleic acid into target DNA sequences.

At present, two classes of transposons are known, i.e. class I and classII transposons.

Class I transposons, also called retrotransposons or retroposons,include retroviral-like retrotransposons and non-retroviral-likeretrotransposons. They work by copying themselves and pasting copiesback into the genome in multiple places. Initially, retrotransposonscopy themselves to RNA (transcription) but, instead of being translated,the RNA is copied into DNA by a reverse transcriptase (often coded bythe transposon itself) and inserted back into the genome. Typicalrepresentatives of class I transposons include e.g. Copia (Drosophila),Ty1 (yeast), THE-1 (human), Bs1 (maize), the F-element, L1 (human) orCin4 (maize).

As a first step Class II transposons have to be transfected to the cellsusing standard methods like virus infection etc. Following that Class IItransposons, also called “DNA-only transposons”, move by a cut and pastemechanism, rather than by copy and paste, and use the transposase enzymein this mechanism. Different types of transposases may work in differentways. Some can bind to any part of the DNA molecule, and the target sitecan be located at any position, while others bind to specific sequences.The transposase then cuts the target site to produce sticky ends,releases the transposon and ligates it into the target site. Typicalclass II representatives include the P element (Drosophila), Ac-Ds(maize), TN3 and IS1 (E. coli), Tam3 (snapdragon) etc.

Particularly, with class II transposons, the element-encoded transposasecatalyzes the excision of the transposon from its original location andpromotes its insertion elsewhere in the genome (Plasterk, 1996 Curr.Top. Microbiol. Immunol. 204, 125-143). Autonomous members of atransposon family can express an active transposase, the transactingfactor for transposition, and thus are capable of transposing on theirown. Non-autonomous elements have mutated transposase genes but mayretain cis-acting DNA sequences. These cis-acting DNA sequences are alsoreferred to as inverted terminal repeats (IR). Some inverted repeatsequences may include one or more direct repeat sequences. Thesesequences usually are embedded in the terminal inverted repeats (IRs) ofthe elements, which are required for mobilization in the presence of acomplementary transposase from another element. Not a single autonomouselement has been isolated from vertebrates so far with the exception ofTol2 (see below); all transposon-like sequences are defective,apparently as a result of a process called “vertical inactivation” (Loheet al., 1995 Mol. Biol. Evol. 12, 62-72). According to one phylogeneticmodel (Hartl et al., 1997 Trends Genet. 13, 197-201), the ratio ofnon-autonomous to autonomous elements in eukaryotic genomes increases asa result of the trans-complementary nature of transposition. Thisprocess leads to a state where the ultimate disappearance of active,transposase-producing copies in a genome is inevitable. Consequently,DNA-transposons can be viewed as transitory components of genomes which,in order to avoid extinction, must find ways to establish themselves ina new host. Indeed, horizontal gene transmission between species isthought to be one of the important processes in the evolution oftransposons (Lohe et al., 1995 supra and Kidwell, 1992. Curr. Opin.Genet Dev. 2, 868-873).

The natural process of horizontal gene transfer can be mimicked underlaboratory conditions. In plants, transposons of the Ac/Ds and Spmfamilies have been routinely transfected into heterologous species(Osborne and Baker, 1995 Curr. Opin. Cell Biol. 7, 406-413). In animals,however, a major obstacle to the transfer of an active transposon systemfrom one species to another has been that of species-specificity oftransposition due to the requirement for factors produced by the naturalhost.

Transposon systems as discussed above may occur in vertebrate andinvertebrate systems. In vertebrates, the discovery of DNA-transposons,mobile elements that move via a DNA intermediate, is relatively recent(Radice, A. D., et al., 1994. Mol. Gen. Genet. 244, 606-612). Sincethen, inactive, highly mutated members of the Tc1/mariner as well as thehAT (hobo/AcTam) superfamilies of eukaryotic transposons have beenisolated from different fish species, Xenopus and human genomes (Oosumiet al., 1995. Nature 378, 873; Ivics et al. 1995. Mol. Gen. Genet. 247,312-322; Koga et al., 1996. Nature 383, 30; Lam et al., 1996. J. Mol.Biol. 257, 359-366 and Lam, W. L., et al. Proc. Natl. Acad Sci. USA 93,10870-10875).

Both invertebrate and vertebrate transposons hold potential fortransgenesis and insertional mutagenesis in model organisms.Particularly, the availability of alternative transposon systems in thesame species opens up new possibilities for genetic analyses. Forexample, piggyBac transposons can be mobilized in Drosophila in thepresence of stably inserted P elements (Hacker et al., (2003), Proc NatlAcad Sci USA 100, 7720-5.). Because P element- and pigyBac-based systemsshow different insertion site preferences (Spradling et al. (1995), ProcNatl Acad Sci USA 92, 10824-30, Hacker et al., (2003), Proc Natl AcadSci USA 100, 7720-5), the number of fly genes that can be insertionallyinactivated by transposons can greatly be increased. P element vectorshave also been used to insert components of the mariner transposon intothe D. melanogaster genome by stable germline transformation. In thesetransgenic flies, mariner transposition can be studied withoutaccidental mobilization of P elements (Lohe and Hartl, (2002), Genetics160, 519-26).

In vertebrates, three active transposons are currently known and used:the Tol2 element in medaka, and the reconstructed transposons SleepingBeauty (SB) and Frog Prince (FP). A further interesting transposonsystem in vertebrates is the PiggyBac transposon system (Ding et al.,Cell, 2005).

The Tol2 element is an active member of the hAT transposon family inmedaka. It was discovered by a recessive mutation causing an albinophenotype of the Japanese medaka (Oryzias latipes), a small freshwaterfish of East Asia. It was found that the mutation is due to a 4.7-kblong TE insertion into the fifth exon of the tyrosinase gene. The DNAsequence of the element, named Tol2, is similar to transposons of thehAT family, including hobo of Drosophila, Ac of maize and Tam3 ofsnapdragon.

Sleeping Beauty (SB) is a Tc1/mariner-like element from fish andexhibits high transpositional activity in a variety of vertebratecultured cell lines, embryonic stem cells and in both somatic and germline cells of the mouse in vivo.

Also Frog Prince (FP) is a Tc1/mariner-like element that was recentlyreactivated from genomic transposon copies of the Northern Leopard Frog(Rana pipiens). An open reading frame trapping method was used toidentify uninterrupted transposase coding regions, and the majority ruleconsensus of these sequences revealed an active transposase gene. Thus,in contrast to the “resurrection” procedure of SB, the relatively youngstate of genomic elements in Rana pipiens made it possible to ground themajority rule consensus on transposon copies derived from a singlespecies. The SB and FP transposons are clearly distinct, sharing only˜50% identity in their transposase sequences.

Transposons as the above, particularly Tol2, SB and FP, as well aspiggyback (Ding et al., Cell 2005), do not interact and thus may be usedas a genetic tool in the presence of others, which considerably broadensthe utility of these elements. The preferences of these transposons toinsert into expressed genes versus non-coding DNA, and preferences forinsertion sites within genes may be substantially different. If so,different patterns of insertion of these transposon systems can beexploited in a complementary fashion. For instance, one could usedifferent transposon systems to transfect several transgenes into cellssequentially, without accidental and unwanted mobilization of alreadyinserted transgenes. In addition, the number of target loci that can bemutagenized by transposon vectors could dramatically increase bycombining different transposon systems in genome-wide screens.

In addition to the variation in transpositional activity in hosts, anddifferences in target site specificity, distinct structural propertiesof various elements could also be advantageous in certain applications.For example, transposon insertions can be utilized to misexpress genesand to look for gain-of-function phenotypes Rorth, P. (1996, A modularmisexpression screen in Drosophila detecting tissue-specific phenotypes.Proc Natl Acad Sci USA 93, 12418-22.) used a modified P elementtransposon that carried an inducible promoter directed out from theelement to force expression of host genes near to transposon insertionsites and detected tissue specific phenotypes. A prerequisite of such anexperimental setup is that the transposon IRs allow read throughtranscription/translation across the IRs.

As was already explained above DNA transposons have been developed asgene transfer vectors in invertebrate model organisms and more recently,in vertebrates too. They also rose to be strong rivals of the retroviralsystems in human gene therapy. As said before the most usefultransposable elements (TEs) for genetic analyses and for therapeuticapproaches are the Class II TEs moving in the host genome via a“cut-and-paste” mechanism (FIG. 1 ), due to their easy laboratoryhandling and controllable nature. Sleeping Beauty (hereinafterabbreviated as “SB”) belongs to the Tc1/mariner family of the“cut-and-paste” transposons. The schematic outline of the transpositionprocess of a “cut-and-paste” TE is represented in FIG. 1 . These mobileDNA elements are simply organized, encoding a transposase protein intheir genome flanked by the inverted terminal repeats (ITR). The ITRscarry the transposase binding sites necessary for transposition (FIG. 1). Their activities can easily be controlled by separating thetransposase source from the transposable DNA harboring the ITRs, therebycreating a non-autonomous TE. In such a two-component system, thetransposon can only move by transsupplementing the transposase protein(FIG. 1 ). Practically any sequence of interest can be positionedbetween the ITR elements according to experimental needs. Thetransposition will result in excision of the element from the vector DNAand subsequent single copy integration into a new sequence environment.

In general the transposon mediated chromosomal entry seems to beadvantageous over viral approaches because on one hand transposons ifcompared to viral systems do not favour so much the active genes and 5′regulatory regions and thus are not so prone to mutagenesis, and on theother hand due to there special mechanism of chromosomal entry into ofthe gene of interest are more physiologically controlled.

SB already proved to be a valuable tool for functional genomics inseveral vertebrate model organisms (Miskey, C., Izsvak, Z., Kawakami, K.and Ivics, Z. (2005); DNA transposons in vertebrate functional genomics.Cell Mol. Life. Sci 62: 629-641) and shows promise for human genetherapeutic applications (Ivics, Z. and Izsvak, Z. (2006). Transposonsfor gene therapy; Curr. Gene Ther. 6: 593-607). However for all of theseapplications the transpositional activity of the system is a key issueof usability and efficiency. Even though functional and valuable ascommonly known and described as of today the transposase activity islikely to be one of the factors that still causes the SB system to reachits limits. Thus, a remarkable improvement of transpositional activitycould breach current experimental barriers in both directions.

Thus, there still remains a need for improving the already valuable SBsystem as a method for introducing DNA into a cell. Accordingly, it isdesired to enhance efficient insertion of transposons of varying sizeinto the nucleic acid of a cell or the insertion of DNA into the genomeof a cell thus allowing more efficient transcription/translation thancurrently available in the state of the art.

The object underlying the present invention is solved by a polypeptideselected from variants of SB10 transposase comprising an amino acidsequence differing from the sequence of native SB10 transposaseaccording to SEQ ID NO: 1 by 1 to 20 amino acids including at least oneof the following mutations or groups of mutations selected from: [0023]K14R, [0024] K13D, [0025] K13A, [0026] K30R, [0027] K33A, [0028] T83A,[0029] I100L, [0030] R115H, R143L, [0032] R147E, [0033]A205K/H207V/K208R/D210E; [0034] H207V/K208R/D210E; [0035]R214D/K215A/E216V/N217Q; [0036] M243H; [0037] M243Q; E267D; [0039]T314N; [0040] G317E.

The “SB10 transposase” is a well-known transposase of the “SleepingBeauty Transposon System”. Its amino acid sequence is included herein asSEQ. ID No. 1 (FIG. 10 ).

“Mutation” or “mutations” is defined herein as the exchange of 1 or moreamino acids of a known amino acid sequence by 1 or more other aminoacids, respectively, and might—if specifically indicated—also a “groupof mutations” or “groups of mutations”. A “group of mutations” or“groups of mutations” are defined herein as the exchange of groups, e.g.3 or 4, of amino acids from the original sequence by 3 or 4 other aminoacids at the indicated positions, respectively. As a definition thefollowing code is used to identify the above mutations. “XNo.Z” meansthat the amino acid “X” of the original amino acid sequence at position“No.” is exchanged for amino acid “Z”, whereas “XNo.Y/X′No.′Z′/X″No.″Z″”is intended to mean that in this mutation amino acids “X” at position“No.”, “X′” in position “No.′” and “X″” in position “No.″” aresimultaneously exchanged for amino acid “Z”, “Z′” and “Z″” respectively.If a “combination of mutations” is defined “//” is used to separate andindicate “simultaneous mutations” in this combination but otherwise isidentical to a single slash “/”.

In a preferred embodiment of the inventive polypeptide it is a variantof SB10 transposase differing from SEQ ID NO: 1 by 1 to 20 amino acidsincluding at least one of the above-listed mutations or groups ofmutations.

The inventive polypeptides (transposase variants), preferably combinedin an inventive transposon as defined below, have several advantagescompared to approaches in the prior art with the most prominentexhibiting a 100 fold increase in the transposase activity if comparedto the activity of natural SB10.

Systematic mutagenesis studies have already been undertaken in the artto increase the activity of the SB transposases like the systematicexchange of the N-terminal 95 AA of the SB transposase for alanine(Yant, S. R., Park, J., Huang, Y., Mikkelsen, J. H G. and Kay, M. A.(2004) Mutational analysis of the N-terminal DNA-binding domain ofSleeping Beauty transposase: critical residues for DNA binding andhyperactivity in mammalian cells. Mol. Cell Biol. 24: 9239-9247). 10 outof these substitutions caused hyperactivity between 200-400% as comparedto SB10 as a reference (Yant, 2004). In addition, a further variantbeing described in the art is SB16 (Baus, J., Liu, L., Heggestad, A. D.,Sanz, S. and Fletcher, B. S. (2005) Hyperactive transposase mutants ofthe Sleeping Beauty transposon. Mol. Therapy 12: 1148-1156), which wasreported to have a 16-fold activity increase as compared to natural SB10and up to now by far the SB transposase published with the highestactivity. SB16 was constructed by combining 5 individual hyperactivemutations (Baus, 2005).

In another preferred embodiment of the inventive polypeptide thevariants are differing by at least 2, or by at least 1 to 8, preferablyby 2 to 7 of the above-listed mutations or groups of mutations, evenmore preferably by at least 4 to 7 of the above-listed mutations orgroups of mutations.

In another preferred embodiment of this inventive polypeptide thevariants of SB10 transposase are selected from variants comprising thefollowing combination of mutations:

-   -   Variant 1: K14R//R214D/K215A/E216V/N217Q;    -   Variant 2: K33A/R115H//R214D/K215A/E216V/N217Q//M243H;    -   Variant 3:        K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 4: K13D/K33A/T83A//H207V/K208R/D210E//M243Q;    -   Variant 5: K13A/K33A//R214D/K215A/E216V/N217Q;    -   Variant 6: K33A/T83A//R214D/K215A/E216V/N217Q//G317E;    -   Variant 7: K14R/T83N/M243Q;    -   Variant 8: K14R/T83A/I100L/M243Q;    -   Variant 9: K14R/T83A/R143L/M243Q;    -   Variant 10: K14R/T83A/R147E/M243Q;    -   Variant 11: K14R/T83A/M243Q/E267D;    -   Variant 12: K14R/T83A/M243Q/T314N;    -   Variant 13:        K14R/K30R/I100L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 14:        K14R/K30R/R143L//A205K/H1207V/K208R/D210E//R214D/K215A/E216V/N217Q/M243H;    -   Variant 15:        K14R/K30R/R147E//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 16:        K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 17:        K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 18:        K14R/K30R/A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/G317E;    -   Variant 19: K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H;    -   Variant 20:        K14R/K30R/R147E//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 21:        K14R/K30R/R143L//A205K4-H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/E2670;    -   Variant 22:        K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215S/E216V/N217Q//M243H/T314N;    -   Variant 23:        K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/G317E;    -   Variant 24:        K14R/K33A/R115H/R143L//R214D/K215A/E216V/N217Q//M243H;    -   Variant 25:        K14R/K33A/R115H/R147E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 26:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 27:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 28:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/G317E;    -   Variant 29: K14R/T83A/M243Q/G317E;    -   Variant 30: K13M/K33A/T83A//R214D/K215A/E216V/N217Q    -   preferably selected from    -   Variant 1: K14R//R214D/K215A/E216V/N217Q;    -   Variant 2: K33A/R115H//R214D/K215A/E216V/N217Q//M243H;    -   Variant 3:        K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 4: K13D/K33A/T83A/H207V/K208R/D210E//M243Q;    -   Variant 5: K13A/K33A//R214D/K215A/E216V/N217Q;    -   Variant 6: K33A/T83A//R214D/K215A/E216V/N217Q//G317E;    -   Variant 7: K14R/T83A/M243Q;    -   Variant 8: K14R/T83A/I100L/M243Q;    -   Variant 9: K14R/T83A/R143L/M243Q;    -   Variant 10: K14R/T83A/R147E/M243Q;    -   Variant 11: K14R/T83N/M243Q/E267D;    -   Variant 12: K14R/T83A/M243Q/T314N;    -   Variant 14:        K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 15:        K14R/K30R/R147E//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 16:        K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 17:        K14R/K30R//A205K/H207V/K208R/D210E/R214D/K215A/E216V/N217Q/M243H/T314N;    -   Variant 18:        K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/G317E;    -   Variant 19: K14R/K33A/R115H//R214D/K215N216V/N217Q//M243H;    -   Variant 20:        K14R/K30R/R147E//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 21:        K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 23:        K14R/K30R/R143L/A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/G317E;    -   Variant 24: K14R/K33A/R115H/R143L/R214D/K215A/E216V/N217Q/M243H;    -   Variant 25:        K14R/K33A/R115H/R147E//R214D/K215A/E216V/N217Q/M243H;    -   Variant 26:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 27:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 28:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/G317E;    -   more preferably selected from    -   Variant 2: K33N/R115H/R214D/K215A/E216V/N217Q//M243H;    -   Variant 3: K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A        E216V/N217Q//M243H;    -   Variant 14:        K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 15:        K14R/K30R/R147E//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 16:        K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 19: K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H;    -   Variant 20:        K14R/K30R/R147E//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 21:        K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 23:        K4R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/G317E;    -   Variant 24:        K14R/K33A/R115H/R143L/R214D/K215A/E216V/N217Q//M243H;    -   Variant 25:        K14R/K33N/R115H/R147E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 26:        K14R/K33N/R115H//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 27:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 28:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/G317E.

These variants of SB10 transposase may also be selected from variantswith combined mutations consisting of the group indicated above,preferably from those wherein the difference of the amino acid sequenceof these variants from native SB10 transposase is consisting of thecombination of mutations indicated above.

In another preferred embodiment of the inventive polypeptide, thevariants of SB10 transposase comprise a sequence of amino acidsdiffering from the amino acid sequence of native SB10 transposaseaccording to SEQ ID NO: 1 by at least the group of mutations [0165]R214D/K215A/E216V/N217Q.

Accordingly, the variants of SB10 transposase which are selected fromvariants comprising the following combination of mutations arepreferred:

-   -   Variant 1: K14R//R214D/K215A/E216V/N217Q;    -   Variant 2: K33A/R115H//R214D/K215A/E216V/N217Q//M243H;    -   Variant 3: K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215N        E216V/N217Q//M243H;    -   Variant 5: K13A/K33A//R214D/K215A/E216V/N217Q;    -   Variant 6: K33A/T83A//R214D/K215A/E216V/N217Q//G317E;    -   Variant 13:        K14R/K30R/I100U/A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 14:        K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 15:        K14R/K30R/R147E//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 16:        K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 17:        K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 18:        K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/G317E;    -   Variant 19: K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H;    -   Variant 20:        K14R/K30R/R147E//A205K/H207V/K208R/D210E//R214D/K215A        E216V/N217Q//M243H/T314N;    -   Variant 21:        K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 22:        K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 23:        K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/G317E;    -   Variant 24:        K14R/K33A/R115H/R143L//R214D/K215A/E216V/N217Q//M243H;    -   Variant 25:        K14R/K33A/R115H/R147E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 26:        K14R/K33N/R115H//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 27:        K14R/K33N/R115H//R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 28:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/G317E;    -   preferably selected from    -   Variant 1: K14R//R214D/K215A/E216V/N217Q;    -   Variant 2: K33A/R115H//R214D/K215A/E216V/N217Q/M243H;    -   Variant 3:        K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 5: K13A/K33A//R214D/K215A/E216V/N217Q;    -   Variant 6: K33A/T83A//R214D/K215A/E216V/N217Q/G317E;    -   Variant 14:        K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 15:        K14R/K30R/R147E//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 16:        K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 17:        K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 18:        K14R/K30R/A205K/H207V/K208R/D210E/R214D/K215A/E216V/N217Q//M243H/G317E;    -   Variant 19: K14R/K33A/R115H/R214D/K215A/E216V/N217Q//M243H;    -   Variant 20:        K14R/K30R/R147E//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 21:        K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 23:        K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/G317E;    -   Variant 24:        K14R/K33A/R115H/R143L//R214D/K215A/E216V/N217Q//M243H;    -   Variant 25:        K14R/K33A/R115H/R147E//R214D/K215A/E216V/N2171Q//M243H;    -   Variant 26:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 27:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 28:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/G317E;    -   more preferably selected from    -   Variant 2: K33A/R115H/R214D/K215A/E216V/N217Q//M243H;    -   Variant 3: K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215N        E216V/N217Q//M243H;    -   Variant 14:        K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 15:        K14R/K30R/R147E//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 16:        K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 19: K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H;    -   Variant 20:        K14R/K30R/R147E//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 21:        K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 23:        K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/G317E;    -   Variant 24:        K14R/K33A/R115H/R143L//R214D/K215A/E216V/N217Q//M243H;    -   Variant 25:        K14R/K33A/R115H/R147E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 26:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 27:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 28:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/G317E.

These variants of SB10 transposase may also be selected from variantswith combined mutations consisting of the group indicated above,preferably from those wherein the difference of the amino acid sequenceof these variants from native SB10 transposase is consisting of thecombination of mutations indicated above.

In another preferred embodiment of the present invention variants ofSB10 transposase comprise a sequence of amino acids differing fromnative SB10 transposase according to SEQ ID NO: 1 by 1 to 20 amino acidsincluding at least the group of mutations and mutation.

-   -   R214D/K215A/E216V/N217Q and    -   K14R.

Accordingly variants of SB10 transposase which are selected fromvariants comprising the following combination of mutations arepreferred:

-   -   Variant 1: K14R//R214D/K215A/E216V/N217Q;    -   Variant 3:        K14R/K30R/A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 13:        K14R/K30R/I100L/A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 14:        K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 15:        K14R/K30R/R147E//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 16:        K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 17:        K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 18:        K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/G317E;    -   Variant 19: K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H;    -   Variant 20:        K14R/K30R/R147E//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 21:        K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 22:        K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 23:        K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/G317E;    -   Variant 24:        K14R/K33A/R115H/R143L//R214D/K215A/E216V/N217Q//M243H;    -   Variant 25:        K14R/K33A/R115H/R147E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 26:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 27: K14R/K33/R115H/R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 28:        K14R/K33A/R115H/R214D/K215A/E216V/N217Q//M243H/G317E;    -   preferably selected from    -   Variant 1: K14R//R214D/K215A/E216V/N217Q;    -   Variant 3:        K14R/K30R//A205K/H207V/K208R/D210E//R214D/K1215A/E216V/N217Q//M243H;    -   Variant 14:        K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 15:        K14R/K30R/R147E//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 16:        K14R/K30R//A205K4-H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 17:        K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q/M243H/T314N;    -   Variant 18:        K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/G317E;    -   Variant 19: K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H;    -   Variant 20:        K14R/K30R/R147E//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 21:        K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 23:        K14R/K30R/R143L//A205K4-H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/T317E;    -   Variant 24:        K14R/K33A/R115H/R143L//R214D/K215A/E216V/N217Q//M243H;    -   Variant 25:        K14R/K33A/R115H/R147E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 26:        K14R/K33A/R115H//R214H/K215A/E216V/N217Q//M243H/E267D;    -   Variant 27:        K14R/K33N/R115H//R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 28:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q/M243H/G317E;    -   more preferably selected from    -   Variant 3:        K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 14:        K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 15:        K14R/K30R/R147E//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 16:        K14R/K30R/A205K/H207V/K208R/D210E//R214/K215A/E216V/N217Q//M243H/E267        D;    -   Variant 19: K14R/K33N/R115H//R214D/K215A/E216V/N217Q//M243H;    -   Variant 20:        K14R/K30R/R147E//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 21:        K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 23:        K14R/K30R/R143L/A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/G317E;    -   Variant 24:        K14R/K33A/R115H/R143L//R214D/K215A/E216V/N217Q//M243H;    -   Variant 25:        K14R/K33A/R115H/R147E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 26:        K14R/K33N/R115H//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 27:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 28:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/G317E.    -   most preferably selected from    -   Variant 19: K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H;    -   Variant 24:        K14R/K33A/R115H/R143L//R214D/K215A/E216V/N217Q//M243H;    -   Variant 25:        K14R/K33A/R115H/R147E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 26:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 27:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 28:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/G317E.

These variants of SB10 transposase may also be selected from variantswith combined mutations consisting of the group indicated above,preferably from those wherein the difference of the amino acid sequenceof these variants from native SB10 transposase is consisting of thecombination of mutations indicated above.

In another very preferred embodiment of the present invention variantsof SB10 transposase comprise a sequence of amino acids differing fromnative SB10 transposase according to SEQ ID NO: 1 by 1 to 20 amino acidsincluding at least the group of mutations and mutation.

-   -   R214D/K215A/E216V/N217Q and    -   K14R,    -   and 2 to 6 additional mutations or groups of mutations selected        from    -   K30R,    -   K33A,    -   R115H,    -   R143L,    -   R147E,    -   A205K/H207V/K208R/D210E;    -   M243H;    -   E267D;    -   T314N;    -   G317E;    -   preferably 3 to 5 additional mutations selected from    -   K30R,    -   K33A,    -   R115H,    -   R143L,    -   R147E,    -   A205K/H207V/K208R/D210E;    -   M243H;    -   E267D;    -   T314N;    -   G317E.

Accordingly variants of SB10 transposase which are selected fromvariants comprising the following combination of mutations arepreferred:

-   -   Variant 3:        K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 14:        K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 15:        K14R/K30R/R147E//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 16:        K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 17:        K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 18:        K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/G317E;    -   Variant 19: K14R/K33A/R115H/R214D/K215A/E216V/N217Q//M243H;    -   Variant 20: K14R/K3        OR/R147E//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 21: K14R/K3        OR/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 23:        K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/T317E;    -   Variant 24:        K14R/K33A/R115H/R143L//R214D/K215A/E216V/N217Q//M243H;    -   Variant 25:        K14R/K33A/R115H/R147E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 26:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 27:        K14R/K33A/R115H/R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 28:        K14R/K33A/R115H//R214A/K215A/E216V/N217Q//M243H/G317E;    -   preferably selected from    -   Variant 14:        K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 19: K14R/K33A/R115H/R214D/K215A/E216V/N217Q//M243H;    -   Variant 20:        K14R/K30R/R147E//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 21:        K14R/K30R/R143L/A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 23:        K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/G317E;    -   Variant 24:        K14R/K33A/R115H/R143L/R214D/K215A/E216V/N217Q//M243H;    -   Variant 25:        K14R/K33A/R115H/R147E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 26:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 27:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 28:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/G317E.

These variants of SB10 transposase may also be selected from variantswith combined mutations consisting of the group indicated above,preferably from those wherein the difference of the amino acid sequenceof these variants from native SB10 transposase is consisting of thecombination of mutations indicated above.

In a highly preferred embodiment of the present invention variants ofSB10 transposase comprise an amino acid sequence differing from thenative SB10 transposase according to SEQ ID NO: 1 by 1 to 20 amino acidsincluding at least the mutations

-   -   R214D/K215A/E216V/N217Q,    -   K14R    -   and 3 to 4 additional mutations selected from    -   K33A,    -   R115H,    -   R143L,    -   R147E,    -   M243H;    -   E267D;    -   T314N;    -   G317E;    -   preferably comprising an amino acid sequence differing from        native SB10 transposase according to SEQ ID No. 1 by 1 to 20        amino acids including at least the group of mutations and        mutations    -   R214D/K215A/E216V/N217Q,    -   K14R    -   K33A,    -   R115H, and    -   M243H;    -   and 0 or 1 additional mutation selected from    -   R143L,    -   R147E,    -   E267D;    -   T314N;    -   G317E.

Accordingly variants of SB10 transposase, which are selected fromvariants comprising the following combination of mutations, arepreferred:

-   -   Variant 19: K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H;    -   Variant 24:        K14R/K33A/R115H/R143L//R214D/K215A/E216V/N217Q//M243H;    -   Variant 25:        K14R/K33A/R115H/R147E//R214D/K215A/E216V/N217Q//M243H;    -   Variant 26:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/E267D;    -   Variant 27:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/T314N;    -   Variant 28:        K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/G317E.

These variants of SB10 transposase may also be selected from variantswith combined mutations consisting of the group indicated above,preferably from those wherein the difference of the amino acid sequenceof these variants from native SB10 transposase is consisting of thecombination of mutations indicated above.

Another aspect of the present invention refers to a nucleic acidcomprising a nucleotide sequence encoding an inventive polypeptide asdefined above. Nucleic acids according to the present inventiontypically comprise ribonucleic acids, including mRNA, DNA, cDNA,chromosomal DNA, extrachromosomal DNA, plasmid DNA, viral DNA or RNA,including also a recombinant viral vector. Thus, in a preferredembodiment of the nucleic acid according to the invention the nucleicacid is DNA or RNA and in another preferred embodiment the nucleic acidis part of a plasmid or a recombinant viral vector. An inventive nucleicacid is preferably selected from any nucleic sequence encoding the aminoacid sequence of the inventive polypeptide. Therefore all nucleic acidvariants coding for the abovementioned inventive mutated SB10 variantsincluding nucleic acid variants with varying nucleotide sequences due tothe degeneration of the genetic code. In particular nucleotide sequencesof nucleic acid variants which lead to an improved expression of theencoded fusion protein in a selected host organism, are preferred.Tables for appropriately adjusting a nucleic acid sequence to the hostcell's specific transcription/translation machinery are known to askilled person. In general, it is preferred to adapt the G/C-content ofthe nucleotide sequence to the specific host cell conditions. Forexpression in human cells an increase of the G/C content by at least10%, more preferred at least 20%, 30%, 50%, 70% and even more preferred90% of the maximum G/C content (coding for the respective inventivepeptide variant) is preferred. Preparation and purification of suchnucleic acids and/or derivatives are usually carried out by standardprocedures (see Sambrook et al. 2001, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor, N.Y.).

These sequence variants preferably lead to inventive polypeptides orproteins selected from variants of SB10 transposase comprising an aminoacid sequence according to SEQ. ID No. 1, which have at least one aminoacid substituted as compared to the native nucleic acid sequence (SEQ.ID. No. 1) of SB10. Therefore, inventive nucleic acid sequences code formodified (non-natural) variants of SB10. Further, promoters or otherexpression control regions can be operably linked with the nucleic acidencoding the inventive polypeptide to regulate expression of thepolypeptide/protein in a quantitative or in a tissue-specific manner.

The inventive polypeptide as defined above can be transfected into acell as a protein or as ribonucleic acid, including mRNA, as DNA, e.g.as extrachromosomal DNA including, but not limited to, episomal DNA, asplasmid DNA, or as viral nucleic acid. Furthermore, the inventivenucleic acid encoding the inventive polypeptide/protein can betransfected into a cell as a nucleic acid vector such as a plasmid, oras a gene expression vector, including a viral vector. Therefore, thenucleic acid can be circular or linear. A vector, as used herein, refersto a plasmid, a viral vector or a cosmid that can incorporate nucleicacid encoding the polypeptide or the transposon (described in moredetail below) of this invention. The terms “coding sequence” or “openreading frame” refer to a region of nucleic acid that can be transcribedand/or translated into a polypeptide in vivo when placed under thecontrol of the appropriate regulatory sequences.

So, in a preferred embodiment of the nucleic acid according to theinvention the nucleic acid additionally comprises at least an openreading frame. In another preferred embodiment, the nucleic acidadditionally comprises at least a regulatory region of a gene.Preferably, the regulatory region is a transcriptional regulatoryregion, and more specifically the regulatory region is selected from thegroup consisting of a promoter, an enhancer, a silencer, a locus-controlregion, and a border element.

DNA encoding the inventive polypeptide can be stably inserted into thegenome of the cell or into a—preferably autonomously replicating—vectorfor constitutive or inducible expression. Where the inventivepolypeptide/protein is transfected into the cell or inserted into thevector as nucleic acid, the inventive polypeptide/protein (an SB10transposase variant) encoding sequence is preferably operably linked toa promoter. There are a variety of promoters that may be used including,but not limited to, constitutive promoters, tissue-specific promoters,inducible promoters, and the like. Promoters are regulatory signals thatbind RNA polymerase in a cell to initiate transcription of a downstream(3′ direction) coding sequence. A DNA sequence is operably linked to anexpression-control sequence, such as a promoter when the expressioncontrol sequence controls and regulates the transcription andtranslation of that DNA sequence. The term “operably linked” includeshaving an appropriate start signal (e.g., ATG) upstream of the DNAsequence to be expressed and maintaining the correct reading frame topermit expression of the DNA sequence under the control of theexpression control sequence to yield production of the desired proteinproduct. In reference to the disclosure above, the inventive DNA or RNAnucleotide sequences may vary even though they code for the sameinventive polypeptide, due to the degeneracy of the three letter codons.For example, it is well known in the art that various specific RNAcodons (corresponding DNA codons, with a T substituted for a U) can beused interchangeably to code for specific amino acids.

Methods for manipulating DNA and proteins are known in the art and areexplained in detail in the literature such as Sambrook et al, (1989)Molecular Cloning: A Laboratory Manual., Cold Spring Harbor LaboratoryPress or Ausubel, R. M., ed. (1994). Current Protocols in MolecularBiology.

In another aspect of the invention refers to an antisense-nucleic acidcomprising a nucleotide sequence which hybridizes under stringentconditions to a nucleic acid according to the invention. This antisenseRNA may be used for silencing purposes. In another embodiment an siRNAis coding for this antisense-RNA according to the invention.

Another aspect of this invention refers to a transposon, also referredto herein as a transposable element, that includes a nucleic acidsequence positioned between at least two repeats (IRs and/or RSDs), atleast one repeat on either side of the nucleic acid sequence.Preferably, the inventive transposon comprises the nucleic acid sequencepositioned between at least two repeats (IRs and/or RSDs) on either sideflanking the nucleic acid sequence in between, wherein these repeats canbind to an inventive polypeptide as defined above and wherein thetransposon is capable of inserting into DNA of a cell, especially iscapable of inserting the nucleic acid sequence or a portion of the intonucleic acid into the DNA of a cell. In other words, repeats are definedas sequences which are recognized and bound by the inventive polypeptideas defined above.

The basic structure of an inventive transposon, which is bound by aninventive polypeptide (a transposase variant), contains a pair of repeatsequences. Therein, the first repeat is typically located upstream tothe above mentioned nucleic acid sequence and the second repeat istypically located downstream of this nucleic acid sequence. In thistypical structure, the second repeat represents the same sequence as thefirst repeat, but shows an inverted reading direction as compared withthe first repeat (5′ and 3′ ends of the complementary double strandsequences are exchanged). These repeats are then termed “invertedrepeats” (IRs), due to the fact that both repeats are just inverselyrepeated sequences.

By another structure repeats as defined above may occur in a multiplenumber upstream and downstream of the above mentioned nucleic acidsequence. Then, preferably two, or eventually three, four, or morerepeats are located upstream and/or downstream to the above mentionednucleic acid sequence. Preferably, the number of repeats locatedupstream and downstream of the above mentioned nucleic acid sequence isidentical. If multiple copies of IRs exist on each terminus of thenucleic acid sequence, some or, more preferably, all of these multiplecopies of the IRs at each terminus may have the same orientation as theIRs and are herein termed “repeats of the same direction” (RSD). In sucha preferred situation the repeat assembly is termed IR/RSD.

For the (IR/RSD) structure, the multiple repeats located upstream and/ordownstream of the above mentioned nucleic acid sequence may be arrangedsuch as to be ligated directly to each other. Alternatively, theserepeats may be separated by a spacer sequence. This spacer sequence istypically formed by a number of nucleic acids, preferably 50 to 200nucleic acids.

The repeats (IRs and/or RSDs) as defined above preferably flank anucleic acid sequence which is inserted into the DNA of a cell. Thenucleic acid sequence can include an open reading frame, especially allor part of an open reading frame of a gene (i.e., the protein codingregion), one or more expression control sequences (i.e., regulatoryregions in nucleic acid) alone or together with all or part of an openreading frame. Preferred expression control sequences include, but arenot limited to promoters, enhancers, border control elements,locus-control regions or silencers. In a preferred embodiment, thenucleic acid sequence comprises a promoter operably linked to at least aportion of an open reading frame. Finally the inventive transposonspreferably occur as a linear transposon (extending from the 5′ end tothe 3′ end, by convention) that can be used as a linear fragment orcircularized, for example in a plasmid.

In one alternative embodiment of the inventive transposon, the nucleicacid sequence positioned between at least two repeats (IRs and/or RSDs)is a nucleic acid according to the invention comprising a nucleotidesequence which encodes an inventive polypeptide. Alternatively, thetransposon may also contain more than one, e.g. 2, 3, or 4, or morecoding regions (regulated under a common promoter and/or individually)for an inventive polypeptide with improved transposase functionality.

In another alternative embodiment of the inventive transposon, thenucleic acid sequence positioned between at least two repeats (IRsand/or RSDs) can be of any recombinant protein. E.g. the protein encodedby the nucleic acid sequence can be a marker protein such as greenfluorescent protein (GFP), the blue fluorescent protein (BFP), the photoactivatable-GFP (PA-GFP), the yellow shifted green fluorescent protein(Yellow GFP), the yellow fluorescent protein (YFP), the enhanced yellowfluorescent protein (EYFP), the cyan fluorescent protein (CFP), theenhanced cyan fluorescent protein (ECFP), the monomeric red fluorescentprotein (mRFP1), the kindling fluorescent protein (KFP1), aequorin, theautofluorescent proteins (AFPs), or the fluorescent proteins IRed,TurboGFP, PhiYFP and PhiYFP-m, tHc-Red (HcRed-Tandem), PS-CFP2 andKFP-Red (all available from EVROGEN, see also www.evrogen.com), or othersuitable fluorescent proteins chloramphenicol acetyltransferase (CAT).The protein further may be selected from “proteins of interest”.Proteins of interest include growth hormones, for example to promotegrowth in a transgenic animal, or from [beta]-galactosidase (lacZ),luciferase (LUC), and insulin-like growth factors (IGFs),α-anti-trypsin, erythropoietin (EPO), factors VIII and XI of the bloodclotting system, LDL-receptor, GATA-1, etc. The nucleic acid sequencefurther may be a suicide gene encoding e.g. apoptotic or apoptoserelated enzymes and genes including AIF, Apaf e.g. Apaf-1, Apaf-2,Apaf-3, or APO-2 (L), APO-3 (L), Apopain, Bad, Bak, Bax, Bcl-2,Bcl-x_(L), Bcl-x_(S), bik, CAD, Calpain, Caspases e.g. Caspase-1,Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7,Caspase-8, Caspase-9, Caspase-10, Caspase-11, or Granzyme B, ced-3,ced-9, Ceramide, c-Jun, c-Myc, CPP32, crm A, Cytochrome c, D4-GDP-DI,Daxx, CdR1, DcR1, DD, DED, DISC, DNA-PKcs, DR3, DR4, DR5, FADD/MORT-1,FAK, Fas, Fas-ligand CD95/fas (receptor), FLICE/MACH, FLIP, Fodrin, fos,G-Actin, Gas-2, Gelsolin, glucocorticoid/glucocorticoid receptor,granzyme A/B, hnRNPs C1/C2, ICAD, ICE, JNK, Lamin A/B, MAP, MCL-1,Mdm-2, MEKK-1, MORT-1, NEDD, NF-κB, NuMa, p53, PAK-2, PARP, Perforin,PITSLRE, PKCS, pRb, Presenilin, prICE, RAIDD, Ras, RIP,Sphingomyelinase, SREBPs, thymidine kinase from Herpes simplex, TNF-α,TNF-α receptor, TRADD, TRAF2, TRAIL-R1, TRAIL-R2, TRAIL-R3,Transglutaminase, U170 kDa snRNP, YAMA, etc. Finally, the nucleic acidsequence being located in the inventive transposon may be selected fromshort RNA hairpin expression cassettes. Also, the nucleic acid sequencemay be either an siRNA (double-stranded RNA of 20 to 25, in particular21 to 23 oligonucleotides, corresponding e.g. to the coding region of agene the expression of which shall be reduced or suppressed or may be anantisense-RNA comprising a nucleotide sequence, which hybridizes understringent conditions to e.g. an mRNA sequence in the cell, therebyreducing or suppressing the translation of the cellular mRNA.

In a further embodiment, the region between the flanking sequence may becomposed of more than one coding regions, e.g. 2, 3, 4 coding regions,which may be mono- or multicistronic. If at least one nucleic acidsequence, which may be involved in therapeutics, diagnostic orscientific applications, is provided to the core of the transposon, atleast one further coding region may code for an inventive polypeptidewith improved transposase functionality.

In general the therapeutic applications of this invention may bemanifold and thus polypeptide, the nucleic acid, and especially thetransposon and/or the gene transfer system according to the inventionmay also find use in therapeutic applications, in which the transposonsystems are employed to stably integrate a therapeutic nucleic acid(“nucleic acid of therapeutic interest”), e.g. gene (nucleic acid oftherapeutic interest), into the genome of a target cell, i.e. genetherapy applications. This may also be of interest for vaccinationtherapy for the integration of antigens into antigen presenting cells,e.g specific tumor antigens, e.g. MAGE-1, for tumor vaccination orpathological antigens for the treatment of infectious diseases derivedfrom pathogens, e.g. leprosy, tetanus, Whooping Cough, Typhoid Fever,Paratyphoid Fever, Cholera, Plague, Tuberculosis, Meningitis, BacterialPneumonia, Anthrax, Botulism, Bacterial Dysentry, Diarrhoea, FoodPoisoning, Syphilis, Gasteroenteritis, Trench Fever, Influenza, ScarletFever, Diphtheria, Gonorrhoea, Toxic Shock Syndrome, Lyme Disease,Typhus Fever, Listeriosis, Peptic Ulcers, and Legionnaires' Disease; forthe treatment of viral infections resulting in e.g. AcquiredImmunodeficiency Syndrome, Adenoviridae Infections, AlphavirusInfections, Arbovirus Infections, Bomrna Disease, BunyaviridaeInfections, Caliciviridae Infections, Chickenpox, Condyloma Acuminata,Coronaviridae Infections, Coxsackievirus Infections, CytomegalovirusInfections, Dengue, DNA Virus Infections, Ecthyma, Contagious,Encephalitis, Arbovirus, Epstein-Barr Virus Infections, ErythemaInfectiosum, Hantavirus Infections, Hemorrhagic Fevers, Viral,Hepatitis, Viral, Human, Herpes Simplex, Herpes Zoster, Herpes ZosterOticus, Herpesviridae Infections, Infectious Mononucleosis, Influenza inBirds, Influenza, Human, Lassa Fever, Measles, Molluscum Contagiosum,Mumps, Paramyxoviridae Infections, Phlebotomus Fever, PolyomavirusInfections, Rabies, Respiratory Syncytial Virus Infections, Rift ValleyFever, RNA Virus Infections, Rubella, Slow Virus Diseases, Smallpox,Subacute Sclerosing Panencephalitis, Tumor Virus Infections, Warts, WestNile Fever, Virus Diseases, Yellow Fever; for the treatment ofprotozoological infections resulting in e.g. malaria. Typically, theantigen used to treat infectious diseases by the inventive transposonsystem contains at least one surface antigen of any bacterial, viral orprotozoological pathogen.

The subject transposon systems may be used to deliver a wide variety oftherapeutic nucleic acids. Therapeutic nucleic acids of interest includegenes that replace defective genes in the target host cell, such asthose responsible for genetic defect based diseased conditions; geneswhich have therapeutic utility in the treatment of cancer; and the like.Specific therapeutic genes for use in the treatment of genetic defectbased disease conditions include genes encoding the following products:factor VIII, factor IX, [beta]-globin, low-density protein receptor,adenosine deaminase, purine nudeoside phosphorylase, sphingomyelinase,glucocerebrosidase, cystic fibrosis transmembrane regulator,[alpha]-antitrypsin, CD-18, omithine transcarbamylase, arginosuccinatesynthetase, phenylalanine hydroxylase, branched-chain [alpha]-ketoaciddehydrogenase, fumarylacetoacetate hydrolase, glucose 6-phosphatase,[alpha]-L-fucosidase, [beta]-glucuronidase, [alpha]-L-iduronidase,galactose 1-phosphate uridyltransferase, and the like. Cancertherapeutic genes that may be delivered via the subject methods include:genes that enhance the antitumor activity of lymphocytes, genes whoseexpression product enhances the immunogenicity of tumor cells, tumorsuppressor genes, toxin genes, suicide genes, multiple-drug resistancegenes, antisense sequences, and the like. The subject methods can beused to not only introduce a therapeutic gene of interest, but also anyexpression regulatory elements, such as promoters, and the like, whichmay be desired so as to obtain the desired temporal and spatialexpression of the therapeutic gene. An important feature of the subjectmethods, the gene therapy application, as described supra, is that thesubject methods may be used for in vivo gene therapy applications. By invivo gene therapy applications is meant that the target cell or cells inwhich expression of the therapeutic gene is desired are not removed fromthe host prior to contact with the transposon system. In contrast,vectors that include the transposon system are administered directly,preferably by injection, to the multicellular organism and are taken upby the target cells, following which integration of the gene into thetarget cell genome occurs.

In a preferred embodiment in the transposon according to the inventionthe nucleic acid sequence is a nucleic acid sequence according to theinvention and/or a nucleic acid sequence coding for a marker proteinsuch as green fluorescent protein (GFP), chloramphenicolacetyltransferase (CAT), a growth hormone, [beta]-galactosidase (lacZ),luciferase (LUC), or insulin-like growth factor (IGFs) and/or a nucleicacid of therapeutic or diagnostic interest.

In a further preferred embodiment, the inventive transposon may occur ina so called “sandwich structure”. By this “sandwich structure” theinventive transposon occurs in two copies flanking a (additional) geneof interest (being located between these two transposons). The geneflanked by the two transposons may directly be linked to thetransposon(s). Alternatively, the active gene(s) may be separated by aspacer sequence from the transposon(s). This spacer sequence istypically formed by a number of nucleic acids, preferably 50 to 200nucleic acids. Furthermore, the proteins or genes encoded by the twotransposons, forming the sandwich structure, may be the same ordifferent. When combining such a “sandwich structure” with an inventivepolypeptide preferably the entire sequence starting from the firsttransposon until the end of the second transposon, will be inserted intoa target (insertion) site of the inventive polypeptide.

In further embodiments thus

the transposon according to the invention thus is part of a plasmid;

in the transposon according to the invention the nucleic acid sequencecomprises an open reading frame;

in the transposon according to the invention the nucleic acid sequencecomprises at least one expression control region; preferably theexpression control region is selected from the group consisting of apromoter, an enhancer or a silencer;

in the transposon according to the invention the nucleic acid sequencecomprises a promoter operably linked to at least a portion of an openreading frame;

the transposon according to the invention is part of a cell obtainedfrom an animal, preferably from a vertebrate or an invertebrate, withpreferably the vertebrate being selected from the group consisting of afish, a bird, or a mammal, preferably a mammal;the transposon according to the invention is integrated into the cellDNA selected from the group consisting of the cell genome orextrachromosomal cell DNA (selected from the group consisting of anepisome or a plasmid) or is autonomously replicated as part of anautonomous vector, e.g. plasmid; and/orin the transposon according to the invention at least one of the repeatscomprises at least one direct repeat.

Another embodiment of the present invention refers to a gene transfersystem. As mentioned above, the inventive polypeptide preferablyrecognizes repeats (IRs and/or RSDs) on the inventive transposon. Thegene transfer system of this invention, therefore, preferably comprisestwo components: the inventive polypeptide (the respective transposase)as defined above and a cloned, non-autonomous (i.e., non-self inserting)element or transposon (referred to herein as a transposon having atleast two repeats (IRs and/or RSDs)) that encompasses between therepeats (IRs and/or RSDs) the transposon substrate DNA. When puttogether these two components of the inventive gene transfer systemprovide active transposon activity and allow the transposon (preferablywith a gene or a sequence of interest with therapeutic, scientific ordiagnostic application) to be relocated. In use, the inventivepolypeptide (transposase variants) binds to the repeats (IRs and/orRSDs) of an inventive transposon and promotes insertion of theintervening nucleic acid sequence into DNA of a cell as defined below.More precisely, the inventive gene transfer system comprises aninventive transposon as defined above in combination with a polypeptideaccording to the invention (or nucleic acid encoding the inventivepolypeptide to provide a transposase activity in a cell). Such aninventive combination preferably results in the insertion of the nucleicacid sequence into the DNA of the cell. Alternatively, it is possible toinsert the transposon of the present invention into DNA of a cellthrough non-homologous recombination through a variety of reproduciblemechanisms. In either event the inventive transposon can be used forgene transfer by using the inventive system.

Thus a further aspect of the invention refers to a gene transfer systemfor introducing DNA into the DNA of a cell comprising:

-   -   a) a transposon according to the invention; and    -   b) a polypeptide according to the invention and/or a nucleic        acid according to the invention (thus encoding the polypeptide        according to the invention); and/or a transposon of the        invention containing a coding region for a polypeptide of the        invention with improved transposase activity.

In another embodiment of the gene transfer system, being an autonomoussystem, the nucleic acid positioned between the at least two repeats inthe transposon according to the invention, comprises the nucleic acidaccording to the invention encoding the polypeptide according to theinvention and e.g. a nucleic acid of interest.

The inventive gene transfer system mediates insertion of the inventivetransposon into the DNA of a variety of cell types and a variety ofspecies by using the inventive polypeptide. Preferably, such cellsinclude any cell suitable in the present context, including but notlimited to animal cells or cells from bacteria, fungi (e.g., yeast,etc.) or plants. Preferred animal cells can be vertebrate orinvertebrate. Preferred invertebrate cells include cells derived fromcrustaceans or mollusks including, but not limited to shrimp, scallops,lobster, claims, or oysters. Preferred vertebrate cells include cellsfrom fish, birds and other animals, e.g. and preferably cells frommammals including, but not limited to, rodents, such as rats or mice,ungulates, such as cows or goats, sheep, swine or cells from a human,preferably rats, mice or humans. In a specifically preferred embodiment,cells suitable for the present invention include CHO, HeLa and COScells.

Furthermore, such cells, particularly cells derived from a mammals asdefined above, can be pluripotent (i.e., a cell whose descendants candifferentiate into several restricted cell types, such as hematopoieticstem cells or other stem cells) and totipotent cells (i.e., a cell whosedescendants can become any cell type in an organism, e.g., embryonicstem cells). Such cells are advantageously used in order to affirmstable expression of the inventive polypeptide (a transposase variant)or to obtain a multiple number of cells already transfected with thecomponents of the inventive gene transfer system. Additionally, cellssuch as oocytes, eggs, and one or more cells of an embryo may also beconsidered as targets for stable transfection with the present genetransfer system.

In another aspect the invention refers to a cell producing the inventivepolypeptide or to a cell containing the inventive nucleic acid ortransposon.

Cells receiving the inventive transposon and/or the inventivepolypeptide/protein and capable of inserting the inventive transposoninto the DNA of that cell also include without being limited thereto,lymphocytes, hepatocytes, neural cells (e.g. neurons, glia cells),muscle cells, a variety of blood cells, and a variety of cells of anorganism, embryonic stem cells, somatic stem cells e.g.(lympho)-hematopoietic cells, embryos, zygotes, sperm cells (some ofwhich are open to be manipulated by an in vitro setting). Morespecifically, the cells derived from the hematopoietic system may be Bcells, T cells, NK cells, dendritic cells, granulocytes, macrophages,platelets, erythrocytes or their (common) progenitor cells, e.g.multipotent progenitor cells, in particular long term or short termCD34+ cells of the hematopoietic system.

In this context, the cell DNA that acts as a recipient of the transposonof this invention includes any DNA present in a cell (as mentionedabove) to be transfected, if the inventive transposon is in contact withan inventive polypeptide within said cell. For example, the cellular DNAcan be part of the cell genome or it can be extrachromosomal, such as anepisome, a plasmid, a circular or linear DNA fragment. Typical targetsfor insertion are e.g. cellular double-stranded DNA molecules.

The components of the inventive gene transfer system, i.e. the inventivepolypeptide/protein (provided in whatever form, e.g. as such (a protein)or encoded by an inventive nucleic acid or as a component of aninventive transposon) and the inventive transposon containing anucleotide sequence (coding for a protein of interest) can betransfected into a cell, preferably into a cell as defined above, andmore preferably into the same cell. Transfection of these components mayfurthermore occur in subsequent order or in parallel. E.g. the inventivepolypeptide, its encoding nucleic acid or a transposon containing theinventive nucleic acid, may be transfected into a cell as defined aboveprior to, simultaneously with or subsequent to transfection of theinventive transposon containing a nucleotide sequence (coding for aprotein of interest). Alternatively, the inventive transposon may betransfected into a cell as defined above prior to, simultaneously withor subsequent to transfection of the inventive polypeptide or itsencoding nucleic acid or a transposon containing the inventive nucleicacid. If transfected parallel, preferably both components are providedin a separated formulation and/or mixed with each other directly priorto administration in order to avoid transposition prior to transfection.Additionally, administration of at least one component of the genetransfer system may occur repeatedly, e.g. by administering at leastone, two or multiple doses of this component, or both components.

For any of the above transfection reactions, the inventive gene transfersystem may be formulated in a suitable manner as known in the art, or asa pharmaceutical composition or kit as defined below.

Furthermore, the components of the inventive gene transfer system arepreferably transfected into one or more cells by techniques such asparticle bombardment, electroporation, microinjection, combining thecomponents with lipid-containing vesicles, such as cationic lipidvesicles, DNA condensing reagents (e.g., calcium phosphate, polylysineor polyethyleneimine), and inserting the components (i.e. the nucleicacids thereof) into a viral vector and contacting the viral vector withthe cell. Where a viral vector is used, the viral vector can include anyof a variety of viral vectors known in the art including viral vectorsselected from the group consisting of a retroviral vector, an adenovirusvector or an adeno-associated viral vector.

As already mentioned above the nucleic acid encoding the inventivepolypeptide may be RNA or DNA. Similarly, either the inventive nucleicacid encoding the inventive polypeptide or the transposon of thisinvention can be transfected into the cell as a linear fragment or as acircularized, isolated fragment or inserted into a vector, preferably asa plasmid or as recombinant viral DNA.

Furthermore, the inventive nucleic acid encoding the inventivepolypeptide/protein or the transposon of the invention is therebypreferably stably or transiently inserted into the genome of the hostcell to facilitate temporary or prolonged expression of the inventivepolypeptide in the cell.

The present invention furthermore provides an efficient method forproducing transgenic animals, including the step of applying theinventive gene transfer system to an animal.

Another embodiment of the present invention refers to a transgenicanimal produced by such methods as disclosed above, preferably by usingthe inventive gene transfer system. Inventive transgenic animalspreferably contain a nucleic acid sequence inserted into the genome ofthe animal by the inventive gene transfer system, thereby enabling thetransgenic animal to produce its gene product, e.g. a protein. Ininventive transgenic animals this protein is preferably a product forisolation from a cell. Therefore, in one alternative, inventivetransgenic animals may be used as bioreactors. The inventive protein canbe produced in quantity in milk, urine, blood or eggs. Promoters can beused that promote expression in milk, urine, blood or eggs and thesepromoters include, but are not limited to, casein promoter, the mouseurinary protein promoter, [beta]-globin promoter and the ovalbuminpromoter respectively. Recombinant growth hormone, recombinant insulin,and a variety of other recombinant proteins have been produced usingother methods for producing protein in a cell. Nucleic acids encodingthese or other proteins can be inserted into the transposon of thisinvention and transfected into a cell. Efficient transfection of theinventive transposon as defined above into the DNA of a cell occurs whenan inventive Polypeptide is present. Where the cell is part of a tissueor part of a transgenic animal, large amounts of recombinant protein canbe obtained.

Inventive transgenic animals may be selected from vertebrates andinvertebrates, selected form e.g. fish, birds, mammals including, butnot limited to, rodents, such as rats or mice, ungulates, such as cowsor goats, sheep, swine or humans.

The present invention furthermore provides a method of treatment for apatient in need thereof by applying the inventive gene transfer system,in which the method is an in-vivo or ex-vivo gene therapy and a nucleicacid of interest is used.

Thus the present invention furthermore provides a method for genetherapy comprising the step of introducing the inventive gene transfersystem into cells as defined above. Therefore, the inventive transposonas defined above preferably comprises a gene to provide a gene therapyto a cell or an organism. Preferably, the gene is placed under thecontrol of a tissue specific promoter or of a ubiquitous promoter or oneor more other expression control regions for the expression of a gene ina cell in need of that gene. A variety of genes are being tested for avariety of gene therapies including, but not limited to, the CFTR genefor cystic fibrosis, adenosine deaminase (ADA) for immune systemdisorders, factors involved in blood clotting, e.g. Factor VII, VIII,factor IX and interleukin-2 (IL-2) for blood cell diseases,alpha-1-antitrypsin for lung disease, and tumor necrosis factors (TNFs).These and a variety of human or animal specific gene sequences includinggene sequences to encode marker proteins and a variety of recombinantproteins are available in the known gene databases such as GenBank, andthe like.

Particularly for gene therapy purposes, but also for other inventivepurposes the inventive gene transfer system may be transfected intocells by a variety of methods, e.g. by microinjection, lipid-mediatedstrategies or by viral-mediated strategies. For example, wheremicroinjection is used, there is very little restraint on the size ofthe intervening sequence of the transposon of this invention. Similarly,lipid-mediated strategies do not have substantial size limitations.However, other strategies for introducing the gene transfer system intoa cell, such as viral-mediated strategies could limit the length of thenucleic acid sequence positioned between the repeats (IRs and/or RSDs),according to this invention.

In this context, the inventive gene transfer system as defined above canbe delivered to cells via viruses, including retroviruses (such aslentiviruses, etc.), adenoviruses, adeno-associated viruses,herpesviruses, and others. There are several potential combinations ofdelivery mechanisms for the inventive transposon portion containing thetransgene of interest flanked by the terminal repeats (IRs and/or RSDs)and the gene encoding the inventive polypeptide (transposase variant).For example, both, the inventive transposon and the inventivepolypeptide (or transposase gene) can be contained together on the samerecombinant viral genome; a single infection delivers both parts of theinventive gene transfer system such that expression of the transposasethen directs cleavage of the transposon from the recombinant viralgenome for subsequent insertion into a cellular chromosome. In anotherexample, the inventive polypeptide (transposase variant) and theinventive transposon can be delivered separately by a combination ofviruses and/or non-viral systems such as lipid-containing reagents. Inthese cases, either the transposon and/or the transposase gene can bedelivered by a recombinant virus. In every case, the expressedtransposase gene directs liberation of the transposon from its carrierDNA (viral genome) for insertion into chromosomal DNA.

In a specific embodiment of the present invention inventive transposonsmay be utilized for insertional mutagenesis, preferably followed byidentification of the mutated gene. DNA transposons, particularly theinventive transposons, have several advantages compared to approaches inthe prior art, e.g. with respect to viral and retroviral methods. Forexample, unlike proviral insertions, transposon insertions can beremobilized by supplying the transposase activity in trans. Thus,instead of performing time-consuming microinjections, it is possibleaccording to the present invention to generate transposon insertions atnew loci by crossing stocks transgenic for the above mentioned twocomponents of the transposon system, the inventive transposon and theinventive polypeptide. In a preferred embodiment the inventive genetransfer system is directed to the germ line of the experimental animalsin order to mutagenize germ cells. Alternatively, transposase expressioncan be directed to particular tissues or organs by using a variety ofspecific promoters. In addition, remobilization of a mutagenictransposon out of its insertion site can be used to isolate revertantsand, if transposon excision is associated with a deletion of flankingDNA, the inventive gene transfer system may be used to generate deletionmutations. Furthermore, since transposons are composed of DNA, and canbe maintained in simple plasmids, inventive transposons and particularlythe use of the inventive gene transfer system is much safer and easierto work with than highly infectious retroviruses. The transposaseactivity can be supplied in the form of DNA, mRNA or protein as definedabove in the desired experimental phase.

When the inventive gene transfer system is used in insertionalmutagenesis screens, inventive transposons preferably comprise fourmajor classes of constructs to identify the mutated genes rapidly, i.e.enhancer traps, promoter traps, polyA traps and gene traps (or exontraps) as defined below. These inventive transposons preferably containa reporter gene, which should be expressed depending on the geneticcontext of the integration.

In enhancer traps, the expression of the reporter typically requires thepresence of a genomic cis-regulator to act on an attenuated promoterwithin the integrated construct.

Promoter traps typically contain no promoter at all. In order to ensureexpression of vectors, the vectors are preferably in-frame in an exon orclose downstream to a promoter of an expressed gene.

In polyA traps, the marker gene preferably lacks a polyA signal, butcontains a splice donor (SD) site. Thus, when integrating into anintron, a fusion transcript can be synthesized comprising the marker andthe downstream exons of the trapped gene.

Gene traps (or exon traps) typically lack promoters, but are equippedwith a splice acceptor (SA) preceding the marker gene. Reporteractivation occurs if the vector is integrated into an expressed gene,and splicing between the reporter and an upstream exon takes place.

Finally, gene trap and polyA trap cassettes can be combined. In thatcase, the marker of the polyA trap part preferably carries a promoter sothat the vector can also trap downstream exons, and both upstream anddownstream fusion transcripts of the trapped gene can be obtained. Theseconstructs also offer the possibility to visualize spatial and temporalexpression patterns of the mutated genes by using LacZ or fluorescentproteins as a marker gene.

In a specific form of the inventive method, the present inventionfurthermore provides an efficient system for gene tagging by introducinga “tag” into a genomic sequence using the inventive gene transfersystem. Any of the above mentioned inventive transposons, e.g. enhancertraps, promoter traps, polyA traps and gene traps (or exon traps), etc.may be used.

Due to their inherent ability to move from one chromosomal location toanother within and between genomes, inventive transposons are suitableas genetic vectors for genetic manipulations in several organisms.Generally, transposon tagging is a technique in which transposons aremobilized to “hop” into genes, thereby inactivating them by insertionalmutagenesis. These methods are discussed e.g. by Evans et al., TIG 199713, 370-374. In the inventive process, the inactivated genes are“tagged” by the transposon which then can be used to recover the mutatedallele. The ability of the human and other genome projects to acquiregene sequence data has outpaced the ability of scientists to ascribebiological function to the new genes. Therefore, the present inventionprovides an efficient method for introducing a tag into the genome of acell. Where the tag is inserted into a location in the cell thatdisrupts expression of a protein that is associated with a particularphenotype, expression of an altered phenotype in a cell containing thenucleic acid of this invention permits the association of a particularphenotype with a particular gene that has been disrupted by thetransposon of this invention. Preferably, the inventive transposon asdefined above functions as a tag. Primers designed to sequence thegenomic DNA flanking the transposon of this invention can be used toobtain sequence information about the disrupted gene.

In a further embodiment the present invention also provides an efficientsystem for gene discovery, e.g. genome mapping, by introducing aninventive transposon as defined above into a gene using the inventivegene transfer system. In one example, the inventive transposon encodinga protein of interest in combination with the inventive Polypeptide or anucleic acid encoding the inventive polypeptide or an inventivetransposon containing the nucleic acid coding for the inventivepolypeptide is transfected into a cell. The transposon—encoding theprotein of interest—preferably comprises a nucleic acid sequencepositioned between at least two repeats (IRs and/or RSDs), wherein therepeats (IRs and/or RSDs) bind to the inventive polypeptide and whereinthe transposon is inserted into the DNA of the cell in the presence ofthe inventive polypeptide. In a preferred embodiment, the nucleic acidsequence includes a marker protein, such as GFP and a restrictionendonuclease recognition site, preferably a 6-base recognition sequence.Following insertion, the cell DNA is isolated and digested with therestriction endonuclease. Where a restriction endonuclease is used thatemploys a 6-base recognition sequence, the cell DNA is cut into about4000-bp fragments on average. These fragments can be either cloned orlinkers can be added to the ends of the digested fragments to providecomplementary sequence for PCR primers. Where linkers are added, PCRreactions are used to amplify fragments using primers from the linkersand primers binding to the direct repeats of the repeats (IRs and/orRSDs) in the transposon. The amplified fragments are then sequenced andthe DNA flanking the direct repeats is used to search computer databasessuch as GenBank.

Using the inventive gene transfer system for methods as disclosed abovesuch as gene discovery and/or gene tagging, permits e.g. the following:

-   1) identification, isolation, and characterization of genes involved    with growth and development through the use of transposons as    insertional mutagens (e.g., see Kaiser et al., 1995, “Eukaryotic    transposons as tools to study gene structure and function.” In    Mobile Genetic Elements, IRL Press, pp. 69-100).-   2) identification, isolation and characterization of transcriptional    regulatory sequences controlling growth and development-   3) use of marker constructs for quantitative trait loci (QTL)    analysis.-   4) identification of genetic loci of economically important traits,    besides those for growth and development, i.e., disease resistance    (e.g., Anderson et al., 1996, Mol. Mar. Biol. Biotech., 5, 105-113).    In one example, the system of this invention can be used to produce    sterile transgenic fish. Broodstock with inactivated genes could be    mated to produce sterile offspring for either biological containment    or for maximizing growth rates in aquacultured fish.

The inventive gene transfer system can also be used as part of a methodfor working with or for screening a library of recombinant sequences,for example, to assess the function of the sequences or to screen forprotein expression, or to assess the effect of a particular protein or aparticular expression control region on a particular cell type. In thisexample, a library of recombinant sequences, such as the product of acombinatorial library or the product of gene shuffling, both techniquesnow known in the art and not the focus of this invention, can beinserted into the transposon of this invention encoding a protein ofinterest to produce a library of transposons with varying nucleic acidsequences positioned between constant repeat sequences (IRs and/orRSDs). The library is then transfected into cells together with theinventive polypeptide as discussed above.

In another embodiment of this invention, the invention provides a methodfor mobilizing a nucleic acid sequence in a cell. According to thismethod the inventive transposon is inserted into DNA of a cell, asdisclosed above. Additionally, the inventive polypeptide, nucleic acidencoding the inventive polypeptide, or a transposon containing thenucleic acid coding for the inventive polypeptide, respectively, istransfected into the cell and the protein is able to mobilize (i.e.move) the transposon from a first position within the DNA of the cell toa second position within the DNA of the cell. The DNA of the cell ispreferably genomic DNA or extrachromosomal DNA. The inventive methodallows movement of the transposon from one location in the genome toanother location in the genome, or for example, from a plasmid in a cellto the genome of that cell.

Additionally, the inventive gene transfer system can also be used aspart of a method involving RNA-interference techniques. RNA interference(RNAi), is a technique in which exogenous, double-stranded RNAs(dsRNAs), being complementary to mRNA's or genes/gene fragments of thecell, are introduced into this cell to specifically bind to a particularmRNA and/or a gene and thereby diminishing or abolishing geneexpression. The technique has proven effective in Drosophila,Caenorhabditis elegans, plants, and recently, in mammalian cellcultures. In order to apply this technique in context with the presentinvention, the inventive transposon preferably contains short hairpinexpression cassettes encoding small interfering RNAs (siRNAs), which arecomplementary to mRNA's and/or genes/gene fragments of the cell. ThesesiRNAs have preferably a length of 20 to 30 nucleic acids, morepreferably a length of 20 to 25 nucleic acids and most preferably alength of 21 to 23 nucleic acids. The siRNA may be directed to any mRNAand/or a gene, that encodes any protein as defined above, e.g. anoncogene. This inventive use, particularly the use of inventivetransposons for integration of siRNA vectors into the host genomeadvantageously provides a long-term expression of siRNA in vitro or invivo and thus enables a long-term silencing of specific gene products.

The present invention further refers to pharmaceutical compositionscontaining either

-   -   an inventive Polypeptide as such or encoded by an inventive        nucleic acid, and/or    -   an inventive transposon; and/or    -   an inventive gene transfer system as defined above comprising an        inventive polypeptide as a protein or encoded by an inventive        nucleic acid, in combination with an inventive transposon.

The pharmaceutical composition may optionally be provided together witha pharmaceutically acceptable carrier, adjuvant or vehicle. In thiscontext, a pharmaceutically acceptable carrier, adjuvant, or vehicleaccording to the invention refers to a non-toxic carrier, adjuvant orvehicle that does not destroy the pharmacological activity of thecomponent(s) with which it is formulated. Pharmaceutically acceptablecarriers, adjuvants or vehicles that may be used in the compositions ofthis invention include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

The pharmaceutical compositions of the present invention may beadministered orally, parenterally, by inhalation spray, topically,rectally, nasally, buccally, vaginally or via an implanted reservoir.

The term parenteral as used herein includes subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrastemal,intrathecal, intrahepatic, intralesional and intracranial injection orinfusion techniques. Preferably, the pharmaceutical compositions areadministered orally, intraperitoneally or intravenously. Sterileinjectable forms of the pharmaceutical compositions of this inventionmay be aqueous or oleaginous suspension. These suspensions may beformulated according to techniques known in the art using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium.

For this purpose, any bland fixed oil may be employed includingsynthetic mono- or di-glycerides. Fatty acids, such as oleic acid andits glyceride derivatives are useful in the preparation of injectables,as are natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, such as carboxymethyl cellulose or similar dispersingagents that are commonly used in the formulation of pharmaceuticallyacceptable dosage forms including emulsions and suspensions. Othercommonly used surfactants, such as Tweens, Spans and other emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms may also be used for the purposes of formulation.

The pharmaceutically acceptable compositions of this invention may beorally administered in any orally acceptable dosage form including, butnot limited to, capsules, tablets, aqueous suspensions or solutions. Inthe case of tablets for oral use, carriers commonly used include lactoseand corn starch. Lubricating agents, such as magnesium stearate, arealso typically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavouring or colouring agents may also be added.

Alternatively, the pharmaceutically acceptable compositions of thisinvention may be administered in the form of suppositories for rectaladministration. These can be prepared by mixing the inventive genetransfer system or components thereof with a suitable non-irritatingexcipient that is solid at room temperature but liquid at rectaltemperature and Therefore will melt in the rectum to release the drug.Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutically acceptable compositions of this invention may alsobe administered topically, especially when the target of treatmentincludes areas or organs readily accessible by topical application,including diseases of the eye, the skin, or the lower intestinal tract.Suitable topical formulations are readily prepared for each of theseareas or organs.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, the pharmaceutically acceptable compositionsmay be formulated in a suitable ointment containing the inventive genetransfer system or components thereof suspended or dissolved in one ormore carriers. Carriers for topical administration of the components ofthis invention include, but are not limited to, mineral oil, liquidpetrolatum, white petrolatum, propylene glycol, polyoxyethylene,polyoxypropylene component, emulsifying wax and water. Alternatively,the pharmaceutically acceptable compositions can be formulated in asuitable lotion or cream containing the active components suspended ordissolved in one or more pharmaceutically acceptable carriers. Suitablecarriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutically acceptable compositions may beformulated as micronized suspensions in isotonic, pH adjusted sterilesaline, or, preferably, as solutions in isotonic, pH adjusted sterilesaline, either with or without a preservative such as benzylalkoniumchloride. Alternatively, for ophthalmic uses, the pharmaceuticallyacceptable compositions may be formulated in an ointment such aspetrolatum.

The pharmaceutically acceptable compositions of this invention may alsobe administered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

The amount of the components of the present invention that may becombined with the carrier materials to produce a composition in a singledosage form will vary depending upon the host treated, the particularmode of administration. It has to be noted that a specific dosage andtreatment regimen for any particular patient will depend upon a varietyof factors, including the activity of the specific component employed,the age, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of a component of the present invention in the composition willalso depend upon the particular component(s) in the composition.

The inventive pharmaceutical composition is preferably suitable for thetreatment of diseases, particular diseases caused by gene defects suchas cystic fibrosis, hypercholesterolemia, hemophilia, e.g. A, B, C orXIII, immune deficiencies including HIV, Huntington disease,α-anti-Trypsin deficiency, as well as cancer selected from colon cancer,melanomas, kidney cancer, lymphoma, acute myeloid leukemia (AML), acutelymphoid leukemia (ALL), chronic myeloid leukemia (CML), chroniclymphocytic leukemia (CLL), gastrointestinal tumors, lung cancer,gliomas, thyroid cancer, mamma carcinomas, prostate tumors, hepatomas,diverse virus-induced tumors such as e.g. papilloma virus inducedcarcinomas (e.g. cervix carcinoma), adeno carcinomas, herpes virusinduced tumors (e.g. Burkitt's lymphoma, EBV induced B cell lymphoma),Hepatitis B induced tumors (Hepato cell carcinomas), HTLV-1 und HTLV-2induced lymphoma, akustikus neurinoma, lungen cancer, pharyngeal cancer,anal carcinoma, glioblastoma, lymphoma, rectum carcinoma, astrocytoma,brain tumors, stomach cancer, retinoblastoma, basalioma, brainmetastases, medullo blastoma, vaginal cancer, pancreatic cancer, testiscancer, melanoma, bladder cancer, Hodgkin syndrome, meningeoma,Schneeberger's disease, bronchial carcinoma, pituitary cancer, mycosisfungoides, gullet cancer, breast cancer, neurinoma, spinalioma,Burkitt's lymphoma, lyryngeal cancer, thymoma, corpus carcinoma, bonecancer, non-Hodgkin lymphoma, urethra cancer, CUP-syndrome,oligodendroglioma, vulva cancer, intestinal cancer, oesphagus carcinoma,small intestine tumors, craniopharyngeoma, ovarial carcinoma, ovariancancer, liver cancer, leukemia, or cancers of the skin or the eye; etc.

The present invention finally refers to kits comprising:

-   -   an inventive polypeptide as such or encoded by an inventive        nucleic acid, and/or    -   an inventive transposon; and/or    -   an inventive gene transfer system as defined above comprising an        inventive polypeptide as such or encoded by an inventive nucleic        acid, in combination with an inventive transposon;        optionally together with a pharmaceutically acceptable carrier,        adjuvant or vehicle, and optional with instructions for use.

Any of the components of the inventive kit may be administered and/ortransfected into cells in a subsequent order or in parallel. E.g. theinventive Polypeptide/protein or its encoding nucleic acid may beadministered and/or transfected into a cell as defined above prior to,simultaneously with or subsequent to administration and/or transfectionof the inventive transposon. Alternatively, the inventive transposon maybe transfected into a cell as defined above prior to, simultaneouslywith or subsequent to transfection of the inventive Polypeptide or itsencoding nucleic acid. If transfected parallel, preferably bothcomponents are provided in a separated formulation and/or mixed witheach other directly prior to administration in order to avoidtransposition prior to transfection. Additionally, administration and/ortransfection of at least one component of the inventive kit may occur ina time staggered mode, e.g. by administering multiple doses of thiscomponent.

All references, patents and publications cited herein are expresslyincorporated by reference into this disclosure. Particular embodimentsof this invention will be discussed in detail and reference has beenmade to possible variations within the scope of this invention. Thereare a variety of alternative techniques and procedures available tothose of skill in the art which would similarly permit one tosuccessfully practice the intended invention.

DESCRIPTION OF FIGURES

FIG. 1 . Scheme of a Class II cut-and-paste transposable element (TE),the binary transposition system created by dissecting the transposasesource from the transposon, and its transposition. ITR, invertedterminal repeat. TEs are moving in the host genome via a “cut-and-paste”mechanism. The mobile DNA elements are simply organized, encoding atransposase protein in their simple genome flanked by the invertedterminal repeats (ITR). The ITRs carry the transposase binding sitesnecessary for transposition. Their activities can easily be controlledby separating the transposase source from the transposable DNA harboringthe ITRs, thereby creating a non-autonomous TE. In such a two-componentsystem, the transposon can only move by transupplementing thetransposase protein. Practically any sequence of interest can bepositioned between the ITR elements according to experimental needs. Thetransposition will result in excision of the element from the vector DNAand subsequent single copy integration into a new sequence environment.

FIG. 2 . A part of the protein alignment of the Tc1 transposasesequences along the whole Tc1 family with no respect of the similarityto SB. (A) Part of the alignment with one picked hyperactive AAsubstitution as an example. (Bari (SEQ ID NO:7); Himar (SEQ ID NO:8);Mosl (SEQ ID NO:9); Impala (SEQ ID NO:10); Minos (SEQ ID NO:11); Tc3(SEQ ID NO:12); Paris (SEQ ID NO:13); S (SEQ ID NO:14); Uhu (SEQ IDNO:15); Quetzal (SEQ ID NO:16); FP (SEQ ID NO:17); SB10 (SEQ ID NO:18);Tc1 (SEQ ID NO:19); Tcb2 (SEQ ID NO:20); Tcb1 (SEQ ID NO:21)). (B)Similarity tree of the alignment.

FIG. 3 . A part of the protein alignment of the Tc1 transposasesequences more related to SB. (A) Part of the alignment with two pickedhyperactive AA substitutions as examples. (Beagle2 (SEQ ID NO:22; PPTN5(SEQ ID NO:23); Froggy2 (SEQ ID NO:24); FP (SEQ ID NO:25); XtTXr2 (SEQID NO: 26); Jumpy2 (SEQ ID NO:27); Maya2 (SEQ ID NO:28); Titof2 (SEQ IDNO:29); Minos2 (SEQ ID NO:30); Xeminosl (SEQ ID NO:31); STURGEON (SEQ IDNO:32); CARMEN (SEQ ID NO:33); Tdrl (SEQ ID NO:34); SB10 (SEQ ID NO:35);XTCons2 (SEQ ID NO:36). (B) Similarity tree of the alignment.

FIG. 4 . Hyperactive mutations forming the base for the shuffling, andtheir grouping to the particular restriction digestions for reducing thewt sequence content.

FIG. 5 . DNaseI treated isolated fragment populations (lane 1 and 2) runon a 12% poly-acrylamide gel. M, marker

FIG. 6 . (A) PCR reassembly reaction (lane 1); M, marker. (B) Final PCRstep for cloning of the full length CDS on the diluted PCR reassemblyreaction template. Lane 1, forward and reverse cloning primers areadded; lane 2, forward cloning primer is added; lane 3, reverse cloningprimer is added; M, marker.

FIG. 7 . Distribution of clone classes in the unselected library1.

FIG. 8 . (A) Mutational participation of the 7 most hyperactive clonesisolated from the shuffling library. (B) Particular statistical featuresof the selected hyperactive clones of the library.

FIG. 9 . Summary of our strategy for the manual improvement of thehyperactive clones harvested from the shuffling library.

FIG. 10 shows the amino acid sequence of SB10 (SEQ ID NO: 1).

FIG. 11 . construction of the vectors used for e.g. the experimentsshown in FIGS. 14 to 26

FIG. 12 . overview of the non-hyperactive transposases; SB10 is the wildtype transposase, SB 11 and SB DNGP are other non-hyperactive orinactive transposases having mutations over SB 10 as indicated and usedherein for comparative reasons. SB10 was originally published by Ivicset al. (1997), Cell 91: 501-510 (FIG. 10 ), while SB 11 was originallypublished by Geurts et al. (2003), Mol. Therapy 8: 108-117. SB 11contains the mutations T136R, M243Q, WA253HVR.

FIG. 13 . overview of the hyperactive mutant transposases SB M3a(containing the mutations K13A, K33A, T83A and R214D/K215A/E216V/N217Qover SB10, Variant 30, table II), SB 3D5-K14R (Variant 19, table II),SB/6A5 (Variant 3, table II), SB 100x (Variant 27, table II), all ofthem derived from SB10. Preferred variants are derived from the sequenceof SB10 with the mutations indicated.

FIG. 14 . comparative analysis of hyperactive transposases SB M3a,SB6/A5 versus non-hyperactive SB10 and SB11 in erythroid lineage

FIG. 15 . comparative analysis of hyperactive transposases M3a, SB6/A5and SB 3D5-K14R in erythroid lineage

FIG. 16 . comparative analysis of hyperactive transposases SB6/A5 andSB100X in erythroid lineage

FIG. 17 . comparative analysis of hyperactive transposases SB6/A5 andSB100X in megakaryotic lineage

FIG. 18 . comparative analysis of hyperactive transposases SB6/A5 andSB100X in granulocyte/macrophage/monocyte lineage

FIG. 19 . relative gene transfer efficiency of transposase SB100X ascompared with the hyperactive transposase SB6/A5

FIG. 20 . relative gene transfer activity of the mutant hyperactivetransposases SB M3a, SB6/A5, SB3D5-K14R and SB100X, compared to wildtype transposase SB10 and the mutant non-hyperactive transposase SB11

FIG. 21 . Stable gene transfer efficiency in human muscleprogenitor/stem cells using mutant hyperactive transposase SB M3a

FIG. 22 . Comparative analysis of gene transfer in human muscleprogenitor/stem cells using mutant hyperactive transposase SB M3a orSB6/A5, compared with wild type transposase SB10 or mutantnon-hyperactive transposase SB11

FIG. 23 . Comparative analysis of hyperactive transposase SB100x, SB3D5-K14R, SB M3a versus non-hyperactive transposase SB11 in muscleprogenitor cells.

FIG. 24 . Levels of Factor IX expression in human muscle progenitorcells following stable transfection using the mutant hyperactivetransposase SB M3a

FIG. 25 . Levels of in vivo expression of Factor IX in liver of miceafter transfection using the mutant hyperactive SB 100X transposase, incomparison with the mutant non-hyperactive SB11 transposase, or aninactive control.

FIG. 26 . Levels of in vivo expression of Factor IX in liver of mice,after transfection using the mutant hyperactive SB 100X transposase,showing a stable expression after partial hepatectomy.

EXAMPLES

Description of the Experimental Strategy

I.) Collecting hyperactive mutations within the SB transposase codingsequence (CDS) for the shuffling.

-   -   a) Mutagenesis through the whole SB CDS.    -   b) Selection of hyperactives using an activity test-system.        II.) In vitro recombination of the selected mutants by DNA        shuffling.    -   a) Isolation of the point mutations on 100-300 bp fragments.    -   b) DNaseI breakage to 30-70 bp fragments.    -   c) PCR shuffling and cloning of the library.    -   d) Sequencing the library.        III.) Searching for clones exhibiting high transpositional        activity.    -   a) Large scale purification of shuffling clones.    -   b) Test of the library clones for transpositional activity in        HeLa cells.    -   c) Manual creation of promising new combinations based on the        sequencing data of the selected hyperactive clones

In all tests for activity as a transposase described here SB10 (Ivics,Z., Hackett, P. B., Plasterk, R. H. and Izsvak, Zs. (1997) Molecularreconstruction of Sleeping Beauty, a Tc1-like transposon from fish, andits transposition in human cells. Cell 91:501-510) was used as acomparator.

I.a) Mutagenesis Through the Whole SB Coding Sequence.

The Tc1 family of transposons is the biggest transposon familyrepresenting a lot of related sequences available for comparison. As afirst step a number of single AA substitutions were designed. A range ofrelated transposase sequences were aligned to find promising positionsto be changed in the SB CDS (coding sequence). Although emphasis waslaid on getting the new AA from known active sequences, also sourceswith no information of their activity and some known inactive sequenceswere used, too. So, the transposase CDSs of other known related Tc1transposones (FIG. 2 .), most of which are coding for activetransposases, were aligned with SB10, followed by a second alignmentwith a range of other Tc1 transposase CDSs more closely related to theSB transposase sequence (compare FIG. 2B and FIG. 3B). FIG. 1A and FIG.2A demonstrate some examples of AA substitution design using the firstand the second alignments respectively.

I.b) Selection of Hyperactives Using an Activity Test-System.

The transpositional activity of all the mutations created was testedusing the classical binary transposition assay (Ivics, 1997, see above).This test was the standard test for transposase activity used here. Thescheme of the two component system is depicted on FIG. 1 . Briefly, HeLacells were cotransfected with the transposon vector carrying theneomycine resistance gene (Neo^(R)) between the SB inverted repeats(pTNeo), and with the Polypeptide (transposase variant) expressingplasmid vector where the expression of the mutant SB transposases wasdriven by the CMV promoter. Following transfection it was selected fortwo weeks with G418 administration for the integration events of theNeo^(R) transposon into the HeLa cells genome. Finally the G418resistant colonies were stained and counted. SB10 transposase CDS wereused as a control to adjust the threshold level of activity and aninactive version of the SB transposase as a negative control. The testswere performed as duplicates on 12 well tissue culture plate formats.

All the polypeptides (all single mutations) according to the invention(transposase variants) causing at least 200% hyperactivity compared toSB10 in the above assay were selected for further use in the shufflingexperiment below. The hyperactivity of these variants was typicallybetween 200-400% compared to SB10.

II.a) Isolation of the Point Mutations on 100-300 bp Fragments.

The PCR shuffling method originally published by Stemmer W. P. C. in1994 (Stemmer, W. P. C. (1994) DNA shuffling by random fragmentation andreassembly: In vitro recombination for molecular evolution. Proc. Natl.Acad. Sci. 91:10747-10751) is a suitable method for mixing relatedparental. All the hyperactive mutations on smaller parts of thetransposase CDS were isolated. The isolated fragments were broken to a30-70 bp fragment population by DNaseI to facilitate high recombinationrates.

41 single hyperactive mutations were collected to combine them in DNAshuffling (FIG. 4 ). The particular mutations on smaller fragments ofthe CDS were isolated using restriction endonucleases to reach higheraverage mutation number/clone. The fragment sizes and the groups ofmutations isolated by the same digestions are summarized on FIG. 4 . Atthe 5′ and 3′ ends of the CDS some extra flanking DNA to the fragmentsincluded to allow rebuilding the full length CDS in the shuffling. Thepredicted average number of mutations pro clone (see FIG. 4 .) wascalculated to be about 4 in the case of this particular library (FIG. 4.).

II.b) DNaseI Breakage to 30-70 bp Fragments

Next the fragments were broken in a random fashion by DNaseI digestions.The similarly sized fragments were treated in groups taking care fortheir same ratio in the total population. Then the mixtures of brokenDNA molecules carrying the mutations were run on 12% acrylamide gels andthe 30-70 bp populations of fragments were isolated. An example of theisolated fragment populations is presented on FIG. 5 .

II.c) PCR Shuffling and Cloning of the Library

The isolated fragment populations were shuffled to reassemble the SBtransposase CDS. Approximately the same amount of all individualmutations was used in the PCR reassembly reaction. As non-overlappingrestriction fragment populations for narrowing the CDS around themutations were used the addition of bridging oligos (for sequence seeconnect1-3 on Table 1) was also necessary to connect the neighboringfragment groups to finally get the full length SB transposase CDS. ThePCR reassembly reaction was done similarly to Stemmer, 1994, (seeabove). Briefly, the isolated 30-70 bp fragment populations of all theselected hyperactive mutations were added in the same ratio into the PCRreaction. The final concentration of DNA in the mixture was about 20ng/μl. Further 2 pmol of each bridging oligos (see Table 1) was added.High-fidelity polymerase was used to minimize the introduction offurther mutations created by the PCR reaction itself. The program forthe PCR reassembly was the following: 1) 94 C°-60 sec, 2) 94 C°-30 sec,3) 50 C°-30 sec, 4) 68 C°-1 min, 5) 68 C°-5 min, and 40 cycles has beenmade of the 2-4 steps. The transposase CDS reassembly to the highermolecular weight was nicely visible after 40 cycles (FIG. 6A.). As thenext step a second PCR reaction was carried out with the SB cloningprimers SBcInfw and SBcInrev (see sequence on Table 1) using the 40×diluted assembly reaction as a template, to amplify the full lengthtransposase CDS. The full length CDS (1023 bp) was amplified using theforward and reverse cloning primers together, in contrast to thesituation when theses were added alone (FIG. 6B). The forward primercarried the recognition site of the endonuclease SpeI. and a Kozaksequence while the reverse one carried an ApaI. recognition site,besides both of the primers beard 26 bp of the very ends of thetransposase CDS. This gave the possibility to efficiently clone theisolated 1023 bp product pool of the second PCR reaction into a suitablevector designed and created for the purposes of the library (data notshown) digested with the same enzymes.

TABLE I Oligonucleotides used for the creation of the shuffling library. Connect1 5′ gtaccacgttcatctgtacaaacaatagtacgcaagtataa 3′ SEQ ID NO: 2 Connect2 5′ cgacataagaaagccagactacggtttgcaactgcacatgggg 3′ SEQ ID NO: 3 Connect3 5′ atattgaagcaacatctcaagacatcagtcaggaagttaaagcttggtcg 3′ SEQ ID NO: 4 SBcInfw5′ ggtcactagtaccatgggaaaatcaaaagaaatcagc ca 3′ SEQ ID NO: 5 SBcInrev5′ ggtcgggcccctagtatttggtagcattgcctttaa  3′ SEQ ID NO: 6II.d) Sequencing the Library.

As a next step the library of the shuffling clones (see IIc) werecharacterized. The library was transferred into E. Coli DH5 competentcells, then isolated and 45 reassembled COS fully sequenced. It wasfound that all the 45 CDS were full length without insertions ordeletions and moreover only a very low incidence of extra mutations wereobserved. Only 2 specific nucleotide positions in the 1023 bp long CDSwere found, where typical point mutations were inserted by the shufflingprocess itself into some of the clones. None of them caused AA change,and they remained silent on the protein level. After aligning thesequences to the SB10 transposase CDS the clonal distribution of the 41mutations taken into the shuffling were identified. The incidence of themutations was fairly statistical in the unselected library, 31 of the 41mutations introduced into the shuffling in the 45 sequenced clones wereidentified (data not shown). However, the average number ofmutations/clone was only about 2 mutations in contrast to the prediction(see above; FIG. 4 .). The reason for this is possibly the 30-70 bplength of the fragments carrying the individual mutations in theshuffling. The majority of the 41 mutations were separated from theirneighboring mutations along the transposase CDS by less then 70 bp longsequences. As a consequence in the shuffling reassembly reaction the30-70 bp fragments could partially exclude each other from a given chainelongation reaction, thereby decreasing the recombination rate betweenthe neighboring mutations. 2 libraries were created with slightmodifications in the shuffling setup (data not shown) and sequenced 23and 22 clones of library 1 and library 2 respectively. Library 1 had 2.2mutations/clone while library 2 had only 1.8, so library 1 was used forthe further experiments. The clonal distribution of mutations in library1 is shown on FIG. 7 .

III.a) Large Scale Purification of Shuffling Clones.

The cell culture system described above was used for the activity tests.A large scale automated purification of plasmid DNA of the shufflingclones was done using a pipetting robot and a plasmid kit. The plasmidpreparations were producing fairly similar yields and their quality wastissue culture compatible. All the plasmid samples were run on agarosegel to verify their similar concentrations and quality. Plasmid DNA ofabout 2000 clones was purified.

III.b) Test of the Library Clones for Transpositional Activity in HeLaCells.

The clones were tested in transposition assays in HeLa cells asdescribed in Ib) above with the difference that 96 well formats wereused. All the tests were done as duplicates. For reference SB16 (Baus,2005) was used on all the plates. All the clones that showed similar orhigher activity compared to SB16 on the duplicated 96 well test plateswere chosen for further operations. Further the activity of the best 20clones on 12 well formats were verified. 7 (Variants 1 to 7) of the 20retested clones showed clearly higher activity compared to SB16. Thebest 2 clones (Variants 2 and 3) exhibited about 2 times higher activitythan SB16 which means about 30 times higher activity compared to SB10.

III.c) Manual Creation of Promising New Combinations Based on theSequencing Data of the 25 Selected Hyperactive Clones

The best 20 clones retested on 12 well format and also 18 other clonesstill showing high activity in the range of SB16, (thus 38 clones alltogether), were fully sequenced to collect a data pool. The mutationalcontent of the best 7 clones is shown in FIG. 8A. The combinations 3D5and 6A5 (variants 2 and 3) proved to be the best showing 30-32 timeshigher activity compared to SB10. By analyzing the sequencing data poolof all 38 active clones it was observed that (i) the mutationnumber/clone is growing with the activity and it reaches the 3, 6mutations/clone as average in the group of the most hyperactive 7 clones(FIG. 8B). Moreover, (ii) it was also realized that the incidence ofsome of the mutations is increasing among clone groups parallel toincreased transpositional activity of the groups. The most obviousexample for this was the increasing incidence of the 214DAVQ (SEQ ID NO:37) mutation (FIG. 8B). Moreover, this particular mutation appeared asthe core mutation of most of the hyperactive combinations reaching orexceeding the activity range of SB16.

Among the 38 sequenced hyperactive clones only 8 containing 4 mutationsand 5 containing 5 mutations were found. This means 21% and 13%incidence of 4 and 5 mutation carrying clones, respectively, in theselected library. No clones were identified bearing more than 5mutations. Moreover, among the 7 best variants already 4 carried 4 or 5mutations (see FIG. 8 ). In the unselected library the incidence ofclones having 4 mutations was less then 10%. Thus, the hyperactivity inthe range of SB16 really correlates well with bearing 4 or 5mutations/clone.

After analyzing the sequencing data of the 38 most hyperactive shufflingclones 3 clones were chosen for further mutagenesis: 3D5, 6A5 and 1281(variants 2, 3 and 7) (FIG. 9 ). Also based on the sequencing data 6“friendly” mutations were identified (FIG. 9 ) with the hope tosuccessfully combining them to the 3 chosen clones. The resultantcombinations and their transpositional activity were measured on 12 weltformats are shown on FIG. 9 and listed in Table 11. In addition, aparticular clone (variant 1) was exceptionally bearing only 2 mutations.Two of these clones were identical (see 2G6 and 6G2 on FIG. 8 ). Thisexceptional combination of the K14R and the 214DAVQ (SEQ ID NO: 37)mutations was obviously not simply additive in terms of hyperactivitybut it was rather a multiplier combination. Based on this observationthe K14R mutation was introduced into the best 3D5 combination, by whichthe resultant clone containing both mutations K14R and 214 DAVQ (SEQ IDNO: 37). The established clone (variant 19) showed highly enhancedactivity (FIG. 9 ).

Overview of Transposase Activity of Tested Variants (Table II)

TABLE II Activity compared Variant (with mutation pattern) to SB10(factor) Variant 1: K14R/R214D//K215A/E216V/N217Q; ~20 Variant 2:K33A/R115H//R214D/K215A/E216V/ ~30 N217Q//M243H; Variant 3:K14R/K30R//A205K/H207V/K208R/ ~30 D210E//R214D/K215A/E216V/N217Q//M243H; Variant 4: K13D/K33A/T83A//H207V/K208R/~20 D210E//M243Q; Variant 5: K13A/K33A//R214D/K215A/E216V/ ~20 N217Q;Variant 6: K33A/T83A//R214D/K215A/E216V/ ~20 N217Q//G317E; Variant 7:K14R/T83A/M243Q; ~15 Variant 8: K14R/T83A/I100L/M243Q; ~5 Variant 9:K14R/T83A/R143L/M243Q; 20-30 Variant 10: K14R/T83A/R147E/M243Q; 20-30Variant 11: K14R/T83A/M243Q/E267D; 15-20 Variant 12:K14R/T83A/M243Q/T314N; ~10 Variant 13: K14R/K30R/I100L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q// M243H; Variant 14:K14R/K30R/R143L//A205K/H207V/ ~40 K208R/D210E//R214D/K215A/E216V/N217Q//M243H; Variant 15: K14R/K30R/R147E//A205K/H207V/ ~30K208R/D210E//R214D/K215A/E216V/N217Q// M243H; Variant 16:K14R/K30R//A205K/H207V/K208R/ ~30 D210E//R214D/K215A/E216V/N217Q//M243H/E267D; Variant 17: K14R/K30R//A205K/H207V/K208R/ ~25D210E//R214D/K215A/E216V/N217Q//M243H/ T314N; Variant 18:K14R/K30R//A205K/H207V/K208R/ ~25 D210E//R214D/K215A/E216V/N217Q//M243H/G317E; Variant 19: K14R/K33A/R115H//R214D/K215A/ 70-80E216V/N217Q//M243H; Variant 20: K14R/K30R/R147E//A205K/H207V/ ~40K208R/D210E//R214D/K215A/E216V/N217Q// M243H/T314N; Variant 21:K14R/K30R/R143L//A205K/H207V/ ~50 K208R/D210E//R214D/K215A/E216V/N217Q//M243H/E267D; Variant 22: K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q// M243H/T314N; Variant 23:K14R/K30R/R143L//A205K/H207V/ ~35 K208R/D210E//R214D/K215A/E216V/N217Q//M243H/G317E; Variant 24: K14R/K33A/R115H/R143L//R214D/ 70-80K215A/E216V/N217Q//M243H; Variant 25: K14R/K33A/R115H/R147E//R214D/70-80 K215A/E216V/N217Q//M243H; Variant 26:K14R/K33A/R115H//R214D/K215A/ 70-80 E216V/N217Q//M243H/E267D; Variant27: K14R/K33A/R115H//R214D/K215A/  90-100 E216V/N217Q//M243H/T314N;Variant 28: K14R/K33A/R115H//R214D/K215A/ 80-90E216V/N217Q//M243H/G317E; Variant 29: K14R/T83A/M243Q/G317E; Variant 30:K13A/K33A/T83A// R214D/K215A/ ~10 E216V/N217Q

Further examples IV to IX were carried out with the object to determinethe activity of various hyperactive transposase mutants as compared tonon-hyperactive or inactive mutants (control experiments) in cell linesof various lineages (see FIGS. 14 to 26 ). The conditions used for theseExamples are given in the following.

Description of the Experimental Strategy

Materials and Methods for Examples IV to IX

A) Sleeping Beauty Transposon System

-   -   The SB transposon system is a binary system composed of (i) the        inverted repeat/direct repeats (IR/DR) flanking the gene of        interest, and (ii) the expression cassette encoding the        transposase. Different transposons containing the gene of        interest and different transposases were used in this study (see        also above).

1) SB Transposon-Based Vectors

pT2-HB-CAG-GFP

The pT2-HB-CAG-GFP transposon is a SB transposon vector in which the GFPreporter gene is transcriptionally regulated by the CAG promotor. TheCAG promotor is a chimeric promoter composed of the CMV (humancytomegalovirus) immediate early enhancer in conjunction with thechicken b-actin/rabbit-b-globin hybrid promoter and intron (CAG)HYPERLINK LMBP 2453); (pA: polyadenylation signal) (FIG. 11 ).

(ii) pT2-HB-CMV-FIX-neo

-   -   The pT2-HB-CMV-FIX-neo transposon is a SB transposon vector in        which the human coagulation factor IX cDNA (FIX) is driven by        the CMV promoter. The vector also contains a Simian Virus 40        (SV40) promoter driving a neomycin resistance gene (Neo^(R))        that confers resistance to G418 (Geneticin) in stably        transfected cells (FIG. 11 ).

(iii) pT2-HB-CMV-GFP-neo

-   -   The pT2-HB-CMV-GFP-neo transposon is a SB transposon vector in        which the GFP reporter gene is transcriptionally regulated by        the CMV promotor. The vector also contains a SV40 promoter        driving a neomycin resistance gene (Neo^(R)) that confers        resistance to G418 (Geneticin) in stably transfected cells (FIG.        11 ).

(iv) pT2-HB-Apo/AAT-FIX

-   -   The pT2-HB-Apo/AAT-FIX transposon is a SB transposon vector in        which the FIX cDNA was driven from the ApoE HCR/AAT promoter        composed of the apolipoprotein E enhancer/al-antitrypsin        promoter, the hepatocyte control region (HCR) and the first FIX        intron (kindly provided by Dr. Miao, University of Washington)        (FIG. 11 ).        2) Transposases

All transposases (active, inactive of hyper-active) are encoded by a CMVexpression plasmid and contain different mutations in the DNA bindingdomain, catalytic domain or both (FIGS. 12 & 13 ) compared to theoriginally reconstructed SB10 Sleeping Beauty transposase. The SB-DNGP(SEQ ID NO: 38) encodes an inactive SB transposase due to the deletionof the DDE catalytic domain. To generate the SB GFP plasmid, the SB100xtransposase (Variant 27) was replaced with GFP.

B) Cells

-   -   Umbilical cord blood (UCB) mononuclear cells were separated from        UCB over Ficoll/Hypaque by centrifugation at 2400 rpm for 30 min        at 20° C., then washed with PBS containing 2 mM EDTA and        centrifuged twice at 1000 rpm for 10 min. The CD34+ cells were        further enriched by immunomagnetic separation according to the        manufacturer's instructions (Miltenyi Biotech Inc. CA, USA)        using magnetic beads conjugated to anti-CD34 antibodies. This        immunomagnetic cell separation typically yielded >95% CD34+        cells which are enriched for hematopoietic stem/progenitor (HSC)        cells.    -   Primary human skeletal muscle stem/progenitors cells (myoblasts)        were obtained by needle biopsy⁵ from the vastus lateralis muscle        of volunteers. Myoblasts were expanded in SkGM medium, as        described by the manufacturer (Cambrex Bio Science, MD USA).        C) Mice    -   C57Bl/6 mice were hydrodynamically transfected with 50        micrograms of transposon with 25 μg of transposase plasmid        diluted in 2 ml of PBS and injected into the tail vein.        Typically, the injection took less than 10 seconds for each        mouse and is results in efficient hepatic gene delivery.        D) Transfection    -   Nucleofection of CD34+ HSCs was done according to the optimized        protocol for human CD34+ cells using the nucleofection kit        developed by Aamaxa Biosystems (Amaxa Biosystems, Cologne        Germany). The U-01 program was employed using the Amaxa        electroporation device (Nucleofector I, Cologne Germany).        Enriched CD34+ cells in PBS were centrifuged at 1200 rpm for 10        min and re-suspended in Nucleofector buffer. Typically, 1.5×10⁵        cells in 100 microliter of human CD34 cell Nucleofector buffer        (Amaxa Biosystems, Cologne Germany) per cuvette were subjected        to electroporation with purified plasmids containing the        transposon (10 microgram) and transposase (5 microgram)        (concentration: 1 microgram/microliter).    -   Nucleofection of human muscle progenitor/stem cells (myoblasts)        was done according to the optimized protocol for human myoblasts        using the nucleofection kit developed by Aamaxa Biosystems        (Amaxa Biosystems, Cologne Germany). The A-33 program was        employed using the Amaxa electroporation device (Nucleofector I,        Cologne Germany). Myoblasts in PBS were centrifuged at 1200 rpm        for 10 min and resuspended in Nucleofector buffer. Typically,        10⁶ cells in 100 microliter of Primary Smooth Muscle Cell        Nucleofector buffer (Amaxa Biosystems, Cologne Germany) per        cuvette were subjected to electroporation with purified plasmids        containing the transposon (3.6 microgram) and transposase (1.4        microgram) (concentration: 1 microgram/microliter). Transfected        myoblasts were selected in G418 (400-600 microgram/ml).        E) Clonogenic Assays

1) CFU-Mk (Megakaryocytes/Platelets)

-   -   Megakaryocytic clonogenic assays were performed by adding 50        microliter of Stemline medium (Sigma-Aldrich, USA) supplemented        with SCF 100 ng/ml, IL-6 20 ng/ml, IL-3 100 ng/ml, FIt3-L 20        ng/ml and TPO 100 ng/ml to the 100 microliter of electroporated        CD34+ cell suspension. Fifty microliter of the final cell        suspension was then added to 450 microliter of megakaryocyte        differentiation medium corresponding to Myelocult H5100        (Stemcell Technologies, Vancouver Canada) supplemented with TPO        25 ng/ml, hSCF 25 ng/ml, hIL-6 10 ng/ml, hIL1b 10 ng/ml and        seeded over 3 wells in a 24-well plate, hence containing 5×10⁴        cells per well. At day 6 post-transfection, medium was changed        by centrifuging the plate briefly, discarding the supernatant        and adding fresh megakaryocyte differentiation medium. At day        10, colonies were counted. GFP expression was monitored using        the Olympus fluorescence inverted microscope and CFU-Mk colonies        were scored with this microscope. In addition, the automated        Zeiss Inverted Microscope was employed.

2) CFU-GM (Granulocyte/Monocyte/Macrophage)

-   -   Granulocyte/monocyte/macrophage clonogenic assays were performed        by adding microliter of the final cell suspension to 270        microliter of granulocyte/monocyte/macrophage differentiation        medium corresponding to semi-solid Methocult GF H4534 (Stemcell        Technologies, Vancouver Canada) composed of 1% methylcellulose        (4000 cps), 30% fetal bovine serum, 1% bovine serum albumin, 10⁴        M 2-mercaptoethanol, 2 mM L-glutamine, 50 ng/ml rhSCF, 10 ng/ml        rhGM-CSF, 10 ng/ml rhIL-3 in Iscove's MDM. The cell suspension        were seeded over 3 wells in a 24-well plate, hence containing        5×10⁴ cells per well. At day 14, colonies were counted. GFP        expression was monitored using the Olympus fluorescence inverted        microscope and CFU-GM colonies were scored with this microscope.        In addition, the automated Zeiss Inverted Microscope was        employed.

3) CFU-E (erythrocytes)

-   -   Erythroid clonogenic assays were performed by adding 30        microliter of the final cell suspension to 270 microliter of        erythoid differentiation medium corresponding to semi-solid        Methocult SF^(BIT) H4436 (Stemcell Technologies, Vancouver        Canada) composed of methylcellulose, fetal bovine serum, bovine        serum albumin, 2-mercaptoethanol, L-glutamine, rhSCF, rhGM-CSF,        rhIL-3, rhIL-6, rhG-CSF, rh Epo in Iscove's MDM. The cell        suspension were seeded over 3 wells in a 24-well plate, hence        containing 5×10⁴ cells per well. At day 7, colonies were counted        which typically contained about 70% glycophorin A⁺ cells, a        characteristic marker of erythroid cells. GFP expression was        monitored using the Olympus fluorescence inverted microscope and        CFU-E colonies were scored with this microscope. In addition the        automated Zeiss Inverted Microscope was employed.

F) Detection of FIX

-   -   The level of FIX in culture supernatant or in citrated plasma        was assayed for FIX antigen by Asserachrome IX sandwich ELISA        (Asserachrome/Diagnostica Stago, Parsippany, N.J., USA). Blood        was collected by retro-orbital bleeds.

G) Microscopy

-   -   Epifluorescence and bright field images were taken with Zeiss        Axiovert 200M microscope, using the Axiovision 4.6 program and        AxioCam MR3 camera. If not mentioned otherwise, pictures were        taken by automatic exposure time selection and optimal display        of the minimum and maximum contained gray or color value (A.        Min/Max option). The settings were kept as same throughout a        series of imaging and were not reset at each individual image.        Confocal microscopy was carried out with Axiovert 100M, LSM510,        Zeiss using the AxioPlan 2 LSM 510 version 2.8 software. In all        images GFP expression was monitored at 488 nm excitation        wavelength.

Examples IV to IX Example IV

Transposition in Human CD34+ Hematopoietic Stem/Progenitor Cells

This Example was intended to provide a comparative analysis ofhyperactive transposases SB M3a and SB6/A5, respectively, versus thenon-hyperactive transposases SB10 and SB11 in erythroid lineage. HumanCD34+ HSC were transfected by nucleofection with the pT2-HB-CAG-GFP andtransposase expression vectors encoding SB M3a and SB 6/A5 as describedin the Materials and Method section for Examples IV to IX. Theperformance of these novel engineered transposases was compared withthat of the originally derived SB10 transposase and SB11. The totalnumber of CFU-E colonies, the absolute number of GFP+ CFU-E colonies andthe % GFP+ CFU-E colonies are shown in FIG. 14 .

The results indicate that transposases SB M3a and SB 6/A5 lead to arobust increase in % GFP+ colonies compared to the originally derivedSB10 transposase and SB11. In contrast, no GFP+ CFU-E colonies weredetectable after co-transfection with the inactive transposase SB DNGPin which the catalytic site had been mutated. Hence, the inventive SBM3a and SB 6/A5 transposases correspond to the inventive group of“hyper-active” transposases that result in more efficient transpositionin human CD34+ HSC compared to non-hyperactive transposase SB10 andSB11. The total number of CFU-E colonies remained unchanged afterelectroporation with the various constructs, suggesting that there is noovert toxicity associated with over-expression of these hyper-activetransposases which underscores the safety of this approach.

Example V

Transposition in Human CD34/Hematopoetic Stem/Progenitor Cells

This comparative analysis was designed to determine the results ofhyperactive transposases M3a, SB6/A5 and SB 3D5-K14R in erythroidlineage. Human CD34+ HSC were transfected by nucleofection with thepT2-HB-CAG-GFP and transposase expression vector encoding SB M3a, SB6/A5 or SB 3D5-K14R, as described above. The total number of CFU-Ecolonies, the absolute number of GFP+ CFU-E colonies and the % GFP+CFU-E colonies are shown in FIG. 15 .

The results indicate that the transposases SB 6/A5 and SB 3D5-K14R leadto a significant increase of 2 and 4.8-fold in % GFP+ relative to thehyperactive transposase SB M3a. In contrast, no GFP+ CFU-E colonies weredetectable after co-transfection with the inactive transposase SB DNGPin which the catalytic site had been mutated. This indicates that the SB3D5-K14R results in even more robust transposition than the hyperactiveSB M3a and SB 6/A5 transposases. Hence, the data shown in FIGS. 14 & 15indicate that SB M3a, SB 6/A5 and SB 3D5-K14R correspond to the group of“hyper-active” transposases that result in more efficient gene transferin human CD34+ HSC compared to non-hyperactive transposases SB10 andSB11. The total number of CFU-E colonies remained unchanged afterelectroporation with the various constructs, suggesting that there is noovert toxicity associated with over-expression of these hyper-activetransposases which underscores the safety of this approach.

Example VI

Transposition in Human CD34+ Hemopoietic Stem/Progenitor Cells

This Example was intended to provide a comparative analysis ofhyperactive transposases SB6/A5 (Variant 3) versus SB100X (Variant 27)in erythroid, megakaryocytic and granulocytic/macrophagemonocyte/lineage. Human CD34+ HSC were transfected by nucleofection withthe pT2-HB-CAG-GFP and transposase expression vector encoding SB 6/A5 orSB 100x, as described above. The total number of CFU-E colonies, theabsolute number of GFP+ CFU-E colonies and the % GFP+ CFU-E colonies areshown in FIG. 16 . The total number of CFU-Mk colonies, the absolutenumber of GFP+ CFU-Mk colonies and the % GFP+ CFU-Mk colonies are shownin FIG. 17 . The total number of CFU-GM colonies, the absolute number ofGFP+ CFU-GM colonies and the % GFP+ CFU-GM colonies are shown in FIG. 18. The % relative increase in % GFP+ CFU-E, CFU-Mk and CFU-GM coloniesfollowing transposition with SB100 vs. SB 6/A5 is shown in FIG. 19 .

The results indicate that the transposases SB 100x (Variant 27) lead toa robust increase in % GFP+ colonies compared to the hyperactivetransposase SB 6/A5 in all lineages (CFU-E, CFU-Mk, CFU-GM). Theincrease in % GFP CFU's, that reflects the concomitant increase instable gene transfer efficiencies following SB-mediated transposition,was consistent among the different lineages and hereby providescompelling evidence that a genuine hematopoietic stem/progenitor cellshad been stable and efficiently transfected using this transposontechnology. In contrast, no GFP+ CFU-E, CFU-Mk and CFU-GM colonies weredetectable after co-transfection with the inactive transposase SB DNGPin which the catalytic site had been mutated. The total number of CFU-E,CFU-Mk and CFU-GM colonies remained unchanged after electroporation withthe various constructs, suggesting that there is no overt toxicityassociated with over-expression of hyper-active transposases SB100x orSB 6/A5.

Comparative analysis of the different transposases in the erythroidlineage indicates that all inventive hyperactive transposases (SB M3a,SB 6A5, SB 3D5-K14R and SB 100x) result in more efficient stable genetransfer in CD34+ HSCs and hence a higher % GFP+ colonies compared towhen the originally derived transposase SB10 and its derivative SB11were used (FIG. 20 ). The SB100x was the most efficient transposaseresulting in ˜100-fold increase in GFP expression and stable genetransfer efficiencies compared to SB10. This is the first demonstrationof such robust stable gene transfer in primary cells, particularlylymphohematopoietic cells, including stem cells, and more in particularhematopoietic stem/progenitor cells using transposon technology. Up tonow, no such high stable gene transfer efficiencies have ever beenreported using a non-viral gene transfer approach in stem cells,particularly in CD34+ HSCs. These data are consistent with a recentdemonstration that only a minor fraction of CD34+ HSCs can be stablytransfected when non-hyperactive transposases are used consistent withthe low % GFP expression in clonogenic assays (Hollis et al. ExpHematol. 2006 October; 34(10): 1333-43) which warrants and justifies thedevelopment of hyper-active transposases as provided herein.

Example VII

Transposition in Human Muscle Stem/Progenitor Cells

This Example was intended to validate inventive hyperactive transposasesSBM3a and SB6/A5. Human muscle stem/progenitor cells (myoblasts) weretransfected by nucleofection with the pT2-HB-CMV-GFP-Neo (see FIG. 11 )and transposase expression vector encoding the hyperactive SB M3atransposase, as described above. Transfected cells were enriched afterG418 selection. High and stable levels of GFP expression were obtainedand most cells survived the G418 selection (FIG. 21 ). In contrast, onlya limited number of GFP+ cells were detectable after cotransfection withthe inactive transposase SB DNGP (SEQ ID NO: 38) in which the catalyticsite had been mutated. These cells ultimately failed to survive the(2418 selection consistent with poor stable gene transfer efficiencies.Comparison of the hyperactive SB M3a transposase with the originallyderived SB10 and its derivative SB11 confirm the superior transpositionefficiency of SB M3a consistent with a robust increase in GFP+transfected cells. Hence, the superior gene transfer efficiencies thatcan be obtained with hyperactive transposases is not unique to a givenprimary cell but can be extended to other cell types, including otherstem/progenitor cells such as muscle stem/progenitor cells (myoblasts).This superior gene transfer potential of inventive hyperactivetransposases translates into efficient and stable production oftherapeutically relevant proteins like human coagulation factor IX (FIG.22 ).

Example VII

Transposition in Human Muscle Stem/Progenitor Cells

This Example serves to validate hyperactive transposases SB 100x, SB3D5-K14R, SB M3a vs. non-hyperactive SB 11. Human muscle stem/progenitorcells (myoblasts) were transfected by nucleofection with thepT2-HB-CMV-GFP-Neo and transposase expression vector encoding thehyperactive SB 100x, SB 3D5-K14R, SB M3a transposase, vs.non-hyperactive SB transposase (SB11) as described above. Transfectedcells were enriched after G418 selection (7 days selection). High andstable levels of GFP expression were obtained and most cells survivedthe G418 selection (FIG. 23 ). In contrast, only a limited number ofGFP+ cells were detectable after cotransfection with the inactivetransposase SB DNGP or SB (“inactive control”) in which the catalyticsite had been mutated. These cells ultimately failed to thrive underG418 selection consistent with poor stable gene transfer efficiencies.The percentage GFP+ cells was limited when the non-hyperactive SB11transposase was used.

Comparison of the hyperactive SB transposases with the SB10-derivativeSB11, confirm the superior transposition efficiency of the SB 100x, SB3D15-K14R and SB M3a, consistent with a robust increase in % GFP+transfected cells. The SB100x transposase yielded the highest % GFP+cells. The stable gene transfer efficiency as reflected by the % GFPcells was less when the SB 3D15 transposase was used relative to SB10x.The stable gene transfer efficiency as reflected by the % GFP cells wasless with the SB M3a transposase compared to SB 3D5-K14R. Hence, therelative differences in transposition/stable gene transfer obtained withdifferent transposases in human muscle progenitor/stem cells correlatedwith the relative differences in gene transfer in other primary celltypes, particularly CD34 human hematopoietic stem/progenitor cells (FIG.20 ). Hence, the superior gene transfer efficiencies that can beobtained with hyperactive transposases is not unique to a given primarycell but can be extended to other cell types, including otherstem/progenitor cells such as muscle stem/progenitor cells (myoblasts).This superior gene transfer potential of hyperactive transposasestranslates into efficient and stable production of therapeuticallyrelevant proteins like human coagulation factor IX followingtransfection with the SB 3D5-K14R transposase and an SB transposoncontaining FIX (FIG. 24 ).

Example IX

Transposition In Vivo

This Example was intended to validate inventive hyperactive transposaseSB100X. To assess whether the hyperactive transposase SB100x alsoresulted in more robust gene transfer in vivo compared to whennon-hyperactive transposases are used, a liver-directed gene transferexperiment was conducted as described above. To achieve this, a plasmidcontaining a transposon expressing factor IX (FIX) from a potentliver-specific promoter (pT2-HB-Apo/AAT-FIX) (see FIG. 11 ) washydrodynamically transfected along with the transposase construct(hyperactive SB 100x vs. non-hyperactive SB 11 vs. inactive SB DNGP orSB GFP) by rapid tail vein injection in C57Bl/6 mice. Stable and hightherapeutic factor IX levels were obtained when the hyperactive SB 100xwas used (FIG. 25 ). In contrast, expression gradually declined when theinactive transposase control was employed (SB GFP). Expression of FIXfollowing co-transfection in vivo of the FIX transposon with thehyper-active SB 100x transposase was also much more robust than when thenon-hyperactive SB 11 transposase was used. Indeed, SB11-mediatedtransposition resulted in FIX expression that gradually declined tolevels slightly above that of the control plasmid that encodes adefective transposase (SB DNGP). These results indicate that prolongedexpression of FIX following SB 100x transfection in vivo could beascribed to efficient stable transposition and hereby confirm thehyper-active transposition properties of SB 100x in vivo.

To confirm that the FIX transposon had been stably integrated into thehepatocyte genome following in vivo gene transfer, hepatocyte cellcycling was induced following partial hepatectomy (Phx) (FIG. 26 ). Thisprocedure consists of surgically removing 60% of the liver. In the weeksfollowing Phx, the liver regenerates by de novo proliferation ofhepatocytes until the normal liver mass had been re-established. SincePhx did not reduce the FIX levels when the SB 100x was used, it providesconclusive evidence that the transgene had integrated into the genome ofthe in viva transfected hepatocytes. In contrast, FIX expressiondeclined in the absence of stable genomic integration followinghydrodynamic co-transfection of the FIX transposon with expressionplasmids that encoded either an inactive transposase (SB-DNGP, inactivecontrol) or no transposase (AAV-MCS).

The invention claimed is:
 1. A transposon comprising: an isolatednucleic acid sequence encoding for a sleeping beauty 10 (SB10)polypeptide variant of SEQ ID NO: 1 positioned between at least tworepeats (inverted repeats (IRs) and/or inverted repeats/direct repeats(IR/DRs)), wherein the repeats can bind to a sleeping beauty 10 (SB10)polypeptide variant of SEQ ID NO: 1, wherein the amino acid sequence ofsaid SB10 polypeptide variant differs from SEQ ID NO: 1 by 1 to 20 aminoacids, wherein one of the mutations is K14R, and wherein the transposoninserts the nucleic acid sequence into the DNA of a mammalian cell. 2.The transposon of claim 1, wherein the nucleic acid sequence comprisesan open reading frame.
 3. The transposon of claim 1, wherein thetransposon is part of a plasmid.
 4. The transposon of claim 1, whereinthe nucleic acid sequence comprises at least one expression controlregion.
 5. The transposon of claim 4, wherein the expression controlregion is selected from the group consisting of: a promoter, an enhancerand a silencer.
 6. The transposon of claim 1, wherein the nucleic acidsequence comprises a promoter operably linked to at least a portion ofan open reading frame.
 7. The transposon of claim 1, wherein the DNA ofthe mammalian cell is selected from the group consisting of: themammalian cell genome or extrachromosomal DNA further selected from thegroup consisting of: an episome and a plasmid.
 8. The transposon ofclaim 1, wherein at least one of the repeats comprises at least onedirect repeat.
 9. A gene transfer system for introducing DNA into DNA ofa mammalian cell comprising the transposon of claim
 1. 10. The genetransfer system of claim 9, wherein the transposon is inserted into thegenome of the mammalian cell.
 11. The gene transfer system of claim 9,wherein the SB10 polypeptide variant comprises a combination ofmutations selected from the group consisting of: Variant 1:K14R/R214D/K215A/E216V/N217Q; Variant 3:K14R/K30R/A205K/H207V/K208R/D210E/R214D/K215A/E216V/N217Q/M243H; Variant7: K14R/T83A/M243Q; Variant 8: K14R/T83A/I100L/M243Q; Variant 9:K14R/T83A/R143L/M243Q; Variant 10: K14R/T83A/R147E/M243Q; Variant 11:K14R/T83A/M243Q/E267D; Variant 12: K14R/T83A/M243Q/T314N; Variant 13:K14R/K30R/I100L/A205K/H207V/K208R/D210E/R214D/K215A/E216V/N217Q/M243H;Variant 14:K14R/K30R/R143L/A205K/H207V/K208R/D210E/R214D/K215A/E216V/N217Q/M243H;Variant 15:K14R/K30R/R147E/A205K/H207V/K208R/D210E/R214D/K215A/E216V/N217Q/M243H;Variant 16:K14R/K30R/A205K/H207V/K208R/D210E/R214D/K215A/E216V/N217Q/M243H/E267D;Variant 17:K14R/K30R/A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q/M243H/T314N;Variant 18:K14R/K30R/A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q/M243H/G317E;Variant 19: K14R/K33A/R115H/R214D/K215A/E216V/N217Q/M243H; Variant 20:K14R/K30R/R147E/A205K/H207V/K208R/D210E/R214D/K215A/E216V/N217Q/M243H/T314N;Variant 21:K14R/K30R/R143L/A205K/H207V/K208R/D210E/R214D/K215A/E216V/N217Q/M243H/E267D;Variant 22:K14R/K30R/R143L/A205K/H207V/K208R/D210E/R214D/K215A/E216V/N217Q/M243H/T314N;Variant 23:K14R/K30R/R143L/A205K/H207V/K208R/D210E/R214D/K215A/E216V/N217Q/M243H/G317E;Variant 24: K14R/K33A/R115H/R143L/R214D/K215A/E216V/N217Q/M243H; Variant25: K14R/K33A/R115H/R147E/R214D/K215A/E216V/N217Q/M243H; Variant 26:K14R/K33A/R115H/R214D/K215A/E216V/N217Q/M243H/E267D; Variant 27:K14R/K33A/R115H/R214D/K215A/E216V/N217Q/M243H/T314N; Variant 28:K14R/K33A/R115H/R214D/K215A/E216V/N217Q/M243H/G317E; and Variant 29:K14R/T83A/M243Q/G317E.
 12. The gene transfer system of claim 9, whereinthe DNA of the mammalian cell is selected from the group consisting of:the mammalian cell genome and extrachromosomal DNA, and further selectedfrom the group consisting of: an episome and a plasmid.
 13. The genetransfer system of claim 9 for introducing DNA into DNA of a mammaliancell, wherein the nucleic acid sequence is transfected into themammalian cell using a method selected from the group consisting of:particle bombardment; electroporation; microinjection; combining thetransposon with lipid-containing vesicles or DNA condensing reagents;and inserting the transposon into a viral vector and contacting theviral vector with the cell.
 14. A mammalian cell comprising thetransposon of claim
 1. 15. An ex vivo or in vivo gene therapy methodcomprising: administering to a subject the gene transfer system of claim9.
 16. The method of claim 15, wherein the gene transfer system isadministered to a mammalian cell selected from the group consisting of:a pluripotent cell, a totipotent cell, a hepatocyte, a neural cell, amuscle cell and a blood hematopoietic system cell.
 17. The method ofclaim 16, wherein the mammalian cell is a cell of the hematopoieticsystem selected from the group consisting of: B cells, T cells, NKcells, dendritic cells, granulocytes, macrophages, platelets, anderythrocytes or their progenitor cells.
 18. A method of producing atransfected target mammalian cell, comprising: providing the genetransfer system of claim 9, providing a target mammalian cell, andtransfecting the target mammalian cell with the gene transfer system.19. A gene transfer system, comprising: a. a polypeptide variant ofsleeping beauty 10 (SB10) transposase comprising an amino acid sequencediffering from the sequence of SB10 transposase SEQ ID NO:1 by 1 to 20amino acids and at least the following mutation: K14R; and b. atransposon comprising a nucleic acid sequence positioned between atleast two repeats (inverted repeats (IRs) and/or inverted repeats/directrepeats (IR/DRs)), wherein the repeats can bind to the SB10 polypeptidevariant, for introducing the nucleic acid sequence into DNA of amammalian cell.
 20. The gene transfer system of claim 19, wherein thenucleic acid sequence comprises at least one open reading frame.
 21. Anex vivo or in vivo gene therapy method comprising: administering to asubject or to cells of a subject the gene transfer system of claim claim19 or
 20. 22. A gene transfer system for introducing a nucleic acidsequence into DNA of a mammalian cell comprising: a. an isolated nucleicacid encoding a variant of sleeping beauty 10 (SB10) transposasecomprising an amino acid sequence differing from the sequence of SB10transposase SEQ ID NO:1 by 1 to 20 amino acids and at least thefollowing mutation: K14R; and b. a transposon comprising an isolatednucleic acid sequence positioned between at least two repeats (invertedrepeats (IRs) and/or inverted repeats/direct repeats (IR/DRs)), whereinthe repeats can bind to the SB10 variant.
 23. The gene transfer systemof claim 22, wherein the nucleic acid sequence comprises at least oneopen reading frame.
 24. An ex vivo or in vivo gene therapy methodcomprising: administering to a subject or to cells of a subject the genetransfer system of claim 22 or
 23. 25. A method of producing atransfected target mammalian cell, comprising: providing the genetransfer system of claim 19, providing a target mammalian cell, andtransfecting the target mammalian cell with the gene transfer system.26. A method of producing a transfected target mammalian cell,comprising: providing the gene transfer system of claim 22, providing atarget mammalian cell, and transfecting the target mammalian cell withthe gene transfer system.
 27. The method of claim 25 or 26, wherein thetransposon is part of a plasmid.
 28. The method of claim 25 or 26,wherein the nucleic acid sequence comprises an open reading frame. 29.The method of claim 25 or 26, wherein the nucleic acid sequence encodesa marker protein.
 30. The method of claim 25 or 26, wherein the nucleicacid sequence comprises at least one expression control region.
 31. Themethod of claim 30, wherein the expression control region is selectedfrom the group consisting of: a promoter, an enhancer and a silencer.32. The method of claim 25 or 26, wherein the nucleic acid sequencecomprises a promoter operably linked to at least a portion of an openreading frame.
 33. The method of claim 25 or 26, wherein the DNA of themammalian cell is selected from the group consisting of: the mammaliancell genome or extrachromosomal DNA further selected from the groupconsisting of: an episome and a plasmid.
 34. The method of claim 25 or26, wherein the transposon is inserted into the genome of the mammaliancell.
 35. The method of claim 25 or 26, wherein at least one of therepeats comprises at least one direct repeat.
 36. The method of claim 25or 26, wherein the SB10 polypeptide variant comprises a combination ofmutations selected from the group consisting of: Variant 1:K14R/R214D/K215A/E216V/N217Q; Variant 3:K14R/K30R/A205K/H207V/K208R/D210E/R214D/K215A/E216V/N217Q/M243H; Variant7: K14R/T83A/M243Q; Variant 8: K14R/T83A/I100L/M243Q; Variant 9:K14R/T83A/R143L/M243Q; Variant 10: K14R/T83A/R147E/M243Q; Variant 11:K14R/T83A/M243Q/E267D; Variant 12: K14R/T83A/M243Q/T314N; Variant 13:K14R/K30R/I100L/A205K/H207V/K208R/D210E/R214D/K215A/E216V/N217Q/M243H;Variant 14:K14R/K30R/R143L/A205K/H207V/K208R/D210E/R214D/K215A/E216V/N217Q/M243H;Variant 15:K14R/K30R/R147E/A205K/H207V/K208R/D210E/R214D/K215A/E216V/N217Q/M243H;Variant 16:K14R/K30R/A205K/H207V/K208R/D210E/R214D/K215A/E216V/N217Q/M243H/E267D;Variant 17:K14R/K30R/A205K/H207V/K208R/D210E/R214D/K215A/E216V/N217Q/M243H/T314N;Variant 18:K14R/K30R/A205K/H207V/K208R/D210E/7R214D/K215A/E216V/N217Q/M243H/G317E;Variant 19: K14R/K33A/R115H/R214D/K215A/E216V/N217Q/M243H; Variant 20:K14R/K30R/R147E/A205K/H207V/K208R/D210E/R214D/K215A/E216V/N217Q/M243H/T314N;Variant 21:K14R/K30R/R143L/A205K/H207V/K208R/D210E/R214D/K215A/E216V/N217Q/M243H/E267D;Variant 22:K14R/K30R/R143L/A205K/H207V/K208R/D210E/R214D/K215A/E216V/N217Q/M243H/T314N;Variant 23:K14R/K30R/R143L/A205K/H207V/K208R/D210E/R214D/K215A/E216V/N217Q/M243H/G317E;Variant 24: K14R/K33A/R115H/R143L/R214D/K215A/E216V/N217Q/M243H; Variant25: K14R/K33A/R115H/R147E/R214D/K215A/E216V/N217Q/M243H; Variant 26:K14R/K33A/R115H/R214D/K215A/E216V/N217Q/M243H/E267D; Variant 27:K14R/K33A/R115H/R214D/K215A/E216V/N217Q/M243H/T314N; Variant 28:K14R/K33A/R115H/R214D/K215A/E216V/N217Q/M243H/G317E; and Variant 29:K14R/T83A/M243Q/G317E.
 37. The method of claim 25 or 26, whereincomponents of the gene transfer system are transfected into themammalian cell using a method selected from the group consisting of:particle bombardment; electroporation; microinjection; combining thetransposon with lipid-containing vesicles or DNA condensing reagents;and inserting the transposon into a viral vector and contacting theviral vector with the cell.
 38. The method of claim 25 or 26, whereinthe gene transfer system is administered to a mammalian cell selectedfrom the group consisting of: a pluripotent cell, a totipotent cell, ahepatocyte, a neural cell, a muscle cell and a blood hematopoieticsystem cell.
 39. The method of claim 25 or 26, wherein the mammaliancell is a cell of the hematopoietic system selected from the groupconsisting of: B cells, T cells, NK cells, dendritic cells,granulocytes, macrophages, platelets, and erythrocytes or theirprogenitor cells.